Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2010 May 19;151(7):3074–3083. doi: 10.1210/en.2010-0049

Regulation of Islet β-Cell Pyruvate Metabolism: Interactions of Prolactin, Glucose, and Dexamethasone

Ramamani Arumugam 1, Eric Horowitz 1, Robert C Noland 1, Danhong Lu 1, Donald Fleenor 1, Michael Freemark 1
PMCID: PMC2903933  PMID: 20484462

Abstract

Prolactin (PRL) induces β-cell proliferation and glucose-stimulated insulin secretion (GSIS) and counteracts the effects of glucocorticoids on insulin production. The mechanisms by which PRL up-regulates GSIS are unknown. We used rat islets and insulinoma (INS-1) cells to explore the interactions of PRL, glucose, and dexamethasone (DEX) in the regulation of β-cell pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), and the pyruvate dehydrogenase kinases (PDKs), which catalyze the phosphorylation and inactivation of PDH. PRL increased GSIS by 37% (P < 0.001) in rat islets. Glucose at supraphysiological concentrations (11 mm) increased PC mRNA in islets; in contrast, PRL suppressed PC mRNA levels in islets and INS-1 cells, whereas DEX was without effect. Neither PRL nor DEX altered PC protein or activity levels. In INS-1 cells, PRL increased PDH activity 1.4- to 2-fold (P < 0.05–0.001) at glucose concentrations ranging from 2.5–11 mm. DEX reduced PDH activity; this effect was reversed by PRL. PDK1, -2, -3, and -4 mRNAs were detected in both islets and insulinoma cells, but the latter expressed trivial amounts of PDK4. PRL reduced PDK2 mRNA and protein levels in rat islets and INS-1 cells and PDK4 mRNA in islets; DEX increased PDK2 mRNA in islets and INS-1 cells; this effect was reversed by PRL. Our findings suggest that PRL induction of GSIS is mediated by increases in β-cell PDH activity; this is facilitated by suppression of PDKs. PRL counteracts the effects of DEX on PDH and PDK expression, suggesting novel roles for the lactogens in the defense against diabetes.

Prolactin induction of glucose-stimulated insulin secretion is mediated by inhibition of pyruvate dehydrogenase kinase expression and consequent increases in pyruvate dehydrogenase activity.

The lactogenic hormones prolactin (PRL) and placental lactogen (PL) induce β cell replication, insulin production, and glucose-stimulated insulin secretion (GSIS) in human fetal pancreas, isolated human islets, and pancreatic islets of newborn and adult rats and mice (1,2,3,4). These effects are mediated through binding to a common PRL receptor, which is expressed in abundance in islet β-cells during late fetal, neonatal, and adult life (5,6,7,8). Constitutive expression of PL in insulinoma cells in vitro and in mouse islets in vivo stimulates β-cell hyperplasia and increases insulin production (9,10). Conversely, targeted knockout of the PRL receptor reduces β-cell mass, GSIS, and glucose tolerance in nonpregnant and pregnant mice (11,12). Thus, the lactogens are essential for normal β-cell development and function.

The mechanisms by which PRL and PL enhance GSIS have not been elucidated. PRL induces β-cell expression of glucose transporter 2 mRNA and glucokinase activity (13,14), suggesting that PRL induction of GSIS may be mediated in part by glucose uptake and use. Indeed, both glucose and PRL induce Forkhead box O (FoxO)-1 phosphorylation, reduce the expression of peroxisome proliferator-activated receptor-α and carnitine palmitoyl transferase-1 mRNAs, and inhibit fatty acid oxidation in islets and insulinoma cells (15,16,17,18). Moreover, both glucose and PRL oppose the effects of glucocorticoids on β-cell proliferation, insulin production, glucokinase activity, and GSIS in insulinoma (INS-1) cells (14,19,20,21,22). However, unlike glucose, PRL is not an acute insulin secretagogue (20,21,22). Moreover, PRL and glucose have synergistic effects on β-cell replication (23), rat insulin gene expression (11,13), and GSIS (20,22). These observations suggest that PRL and glucose exert insulinotropic effects through distinct but overlapping mechanisms of action.

In this study, we compared the effects of PRL and glucose on β-cell metabolism of pyruvate, which is converted to acetyl-CoA by pyruvate dehydrogenase (PDH) or to oxaloacetate by pyruvate carboxylase (PC). PDH is in turn regulated by the pyruvate dehydrogenase kinases (PDKs), which phosphorylate PDH and thereby inhibit its activity. We analyzed the effects of PRL and glucose on expression of PC, PDH, and PDKs in isolated rat islets and rat INS-1 cells and explored the interactions of PRL with dexamethasone (DEX), which inhibits GSIS.

Materials and Methods

Materials

RPMI 1640, DMEM, l-glutamine, antibiotic/antimycotic solution, fetal bovine serum (FBS), and Trizol reagent were purchased from Life Technologies (Rockville, MD). DEX was from Sigma Corp. (St. Louis, MO). Rat PRL (lot AFP7545E) was purchased from Dr. Albert F. Parlow (Hormone Distribution Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA). The Bradford protein reagents were from Pierce Biotechnology (Rockford, IL). Rat insulin RIA Coat-a-Count kits were purchased from Diagnostic Products Corp. (Los Angeles, CA). The high-capacity cDNA archive kits and SYBR Green PCR master mixes were purchased from Applied Biosystems Inc. (Foster City, CA). Radioimmunoprecipitation assay (RIPA) lysis buffer kit for whole-cell lysate protein isolation was from Santa Cruz Biotechnologies (Santa Cruz, CA). A mouse monoclonal antibody to γ-tubulin was from Sigma. Goat polyclonal PDK2 and PDK4 antibodies were from Santa Cruz Biotechnologies.

Cell culture

Primary rat islets were isolated from about 250-g male Wistar rats by a previously described procedure (24). The preincubation medium (used during the first 24 h after isolation) was RPMI 1640 containing 6.8 mm glucose, 10% FBS, 10 mm HEPES, and 1% antibiotic/antimycotic solution. After washing, the islets were incubated for varying periods of time with hormones or diluents in RPMI 1640 basal medium containing 0.5% BSA and varying glucose concentrations. Preliminary experiments showed half-maximal and maximal effects of PRL at concentrations equal to 2 and 20 nm, respectively; these concentrations are comparable with the circulating concentrations of PRL in pregnant women (∼5–8 nm), nonpregnant women (∼0.5–1 nm), and newborn infants (∼8–16 nm) (25).

Rat INS-1 cells (originally supplied by Dr. C. Wollheim, University Medical Center, Geneva, Switzerland) were grown in RPMI 1640 (11.1 mm glucose) supplemented with 10% FBS, 50 μm 2-mercaptoethanol, 1 mm sodium pyruvate, 10 mm HEPES, and 1% antibiotic/antimycotic solution in 5% CO2 at 37 C. At approximately 80% confluence, the cells were washed and incubated with hormones or diluents for varying periods of time in basal medium containing DMEM with 0.1% human serum albumin (HSA), 10 μg/ml human transferrin, 5 μm ethanolamine, 0.1 nm T3, 50 μm phosphoethanolamine, 1% antibiotic/antimycotic solution, and varying concentrations of glucose.

Basal insulin secretion and GSIS

Basal insulin secretion and GSIS were assessed in isolated rat islets. The islets were incubated for 48 h in RPMI 1640 containing 6.8 mm glucose and 10% FBS. The islets were then washed and incubated for 24 h in serum-free basal medium (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL. The islets were washed and preincubated for 1 h in serum-free secretion buffer (114 mm NaCl; 4.7 mm KCl; 1.2 mm KH2PO4; 1.16 mm MgSO4; 2.5 mm CaCl2; 25 mm NaHCO3; 20 mm HEPES; and 0.2% fatty acid free BSA, pH 7.4) containing 2.5 mm glucose. The islets were then transferred to fresh secretion buffer (2.5 mm glucose) and incubated for an additional hour. The conditioned media were collected for measurement of basal insulin secretion. The islets were then transferred to fresh secretion buffer containing 16.7 mm glucose. After an incubation lasting 1 h, the conditioned media were collected for measurement of GSIS. The islets were washed and lysed in RIPA lysis buffer. Insulin was measured with a Coat-a-Count insulin assay kit (Diagnostic Products Corp.) and expressed per microgram of cell protein. Protein was determined by the bicinchoninic acid (BCA) assay (26), using BSA as standard.

Quantification of mRNA expression

Islets and INS-1 cells were washed with serum-free basal media and incubated for 24 h with varying concentrations of glucose in the presence or absence of PRL, DEX, or a combination of the two. Total RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturer’s protocol. cDNA was synthesized from RNA using the high-capacity cDNA archive kit (Applied Biosystems) according to the manufacturer’s protocol. Quantitative real-time PCR (Q-RTPCR) was performed using an ABI 7300 real-time PCR system (Applied Biosystems). Oligonucleotide primers were designed using the Primer Express program from Applied Biosystems. Amplicon lengths ranged from 90 to 150 bp; all primer pairs spanned introns. Negative controls were processed without reverse transcriptase. All samples from a single experiment were run using a single PCR mixture. Expression levels were normalized against the levels of actin or acidic ribosomal phosphoprotein P0 (riboprotein), a housekeeping gene that shows little change during cellular growth or differentiation (27).

The levels of mRNA were quantified using the comparative threshold cycle (CT) method. Table 1 shows the sequences of the primers used for Q-RTPCR and the mean CT values obtained in INS-1 cells and isolated islets (before the change to basal medium).

Table 1.

Analysis of gene expression in rat islets and INS-1 cells by Q-RTPCR

Gene Accession no. Primer sequence (top forward, bottom reverse) (5′–3′) Mean CT islets Mean CT INS-1 cells
Rat PC NM_012744 CGG GCC CAG AAG TTG CT TTG GAG TGG TAG GGC CAT TG 25 23
Rat PDK1 NM_053826 CGC CAC TCT CCA TGA AGC A AAC GAG GTC TTT TCA CAA GCA TT 24 25
Rat PDK2 NM_030872 GTC CCC GCT GTC CAT GAA GTT TTC TCG CAG GCA TTG C 25 26
Rat PDK3 NM_001106581 GCG CCA AGG CCG AGC CCC AG CAG ATC CGC CAA GCC GTC CC 26 24
Rat PDK4 NM_053551 TCA CGG AAT GCC CCT TTG TGG CGT AGA GAC GGG AAA TC 21 36
Open in a new tab

The table shows the oligonucleotide primer pairs, all of which encode rat genes. The mean CT values shown in the table were obtained in islets grown in RPMI 1640 (6.8 mm glucose) supplemented with 10% FBS and in INS-1 cells grown in RPMI 1640 (11.1 mm glucose) supplemented with 10% FBS, 50 μm 2-mercaptoethanol, 1 mm sodium pyruvate, 10 mm HEPES, and 1% antibiotic/antimycotic solution. 

Quantification of PC protein levels by Western blot

For measurements of PC protein levels in islets and INS-1, we loaded protein lysates on denaturing NuPAGE 3–8% Tris-acetate gels (Invitrogen, Carlsbad, CA); the proteins were then transferred to polyvinylidene difluoride membranes (Invitrogen). After blocking overnight in 5% milk diluted in Tris-buffered saline and 0.2% Tween 20 (TBS-T), the membranes were incubated for 45 min with horseradish peroxidase-conjugated streptavidin (Chemicon, Temecula, CA) diluted 1:4000 in TBS-T containing 5% BSA. Membranes were washed for 4–5 min in TBS-T. PC levels were quantified on a VersaDoc model 4000 imaging system (Bio-Rad, Los Angeles, CA), using the biotin-binding protein propionyl-CoA carboxylase as a loading control (Bio-Rad) (28).

PC enzyme activity

Islets and INS-1 cells treated with PRL or diluent were washed in ice-cold PBS three times and dissolved in 250 μl buffer of 250 mm sucrose; 1 mm EDTA; 10 mm Tris-HCl; and 2 mm ATP, pH 7.4 (29,30). The cell lysates were frozen at −80 C before measurements of PC activity.

PC enzyme activity was measured using a slight modification of the method of MacDonald and colleagues (31,32). Forty microliters of cell lysate were incubated at 37 C for 30 min with 160 μl of reaction buffer, which contained 160 mm Tris-HCl; 16.6 mm MgCl2; 166 mm KCl; 13.3 mm sodium pyruvate; 270 mm acetyl CoA; 0.17% Triton X-100, pH 7.6; and 1.6 μCi [14C]NaHCO3/ml. The reaction was terminated by addition of an equal volume of 10% trichloroacetic acid. Eighty percent of the resulting mixture were transferred to scintillation vials and allowed to evaporate to dryness for 20–24 h to allow liberation of unreacted 14CO2. Radioactivity incorporated into resulting dicarboxylic acids was estimated by liquid scintillation spectrometry. Protein was determined by the BCA assay (26) using BSA as standard. The results were expressed as nanomoles per milligram protein per hour.

PDH enzyme activity

PDH activity was measured in islets and INS-1 cells. After a 24-h incubation with PRL or diluent in serum-free basal medium (containing glucose concentrations 2.5, 5.5, and 11 mm), the cells were washed in ice-cold PBS three times, scraped, and lysed in 250 μl buffer of 250 mm sucrose; 1 mm EDTA; 10 mm Tris-HCl; and 2 mm ATP, pH 7.4 (29,30). The cell lysates were frozen at −80 C until oxidation experiments were performed. At the time of the assay, the cell lysates were freeze thawed three times and passed through a 1-ml syringe 10 times.

The liberation of 14CO2 from [1-14C] pyruvate was used to assess PDH activity as previously described (33,34). The reaction buffer contained [1-14C] pyruvate (1 μCi/ml, 1 mmol) in 100 mm sucrose, 10 mm Tris-HCl, 10 mm KPO4, 100 mm KCl, 1 mm MgCl2·6H2O, 1 mm l-carnitine, 0.1 mm malate, 2 mm ATP, 0.05 mm CoA, 1 mm dithiothreitol, and 0.5% BSA (pH 7.4). Forty microliters of the cell lysate were plated in quadruplicate in a modified 48-well cell culture plate (Costar, Cambridge, MA) as described previously (29,30). A small groove was engineered between adjacent wells so that CO2 could freely diffuse between the incubation and trap wells. Reactions were begun by the addition of 160 μl reaction buffer. Culture plates were sealed with Parafilm and a siliconized rubber gasket and allowed to incubate in a shaking water bath at 37 C. Reactions were terminated after 60 min by addition of 100 μl of 70% perchloric acid to the incubation wells. The incubation plate was transferred to an orbital shaker and 14CO2 was trapped in 200 μl of 1 n NaOH for 1 h at room temperature. Trapped radioactivity was determined by liquid scintillation spectrometry. Protein was determined by the BCA assay (26) using BSA as standard. The results were expressed as nanomoles per milligram protein per hour, calculated using the following formula: [(disintegrations per minutesample − disintegrations per minuteblank)/(disintegrations per minute per nanomole)] per milligram protein per hour.

Quantification of PDK2 and PDK4 protein levels by Western blot

Islets were washed with serum-free basal media and incubated with hormones or diluent for 24 h to assess PDK2 and PDK4 protein levels. The islets were washed in ice-cold PBS, collected, and centrifuged at 5000 × g for 2 min. The resulting pellets were then either frozen at −80 C or used immediately for protein extraction. RIPA lysis buffer kit (Santa Cruz Biotechnologies) was used to prepare whole-cell lysates for Western blotting.

For measurement of PDK2 and PDK4 protein, we separated 30 μg of islet protein extracts on 4–12% Bis-Tris SDS-PAGE gels (Invitrogen) and transferred the proteins to polyvinylidene fluoride membranes. The membranes were washed in TBS-T and then blocked in 4% milk buffer (Chemicon) + 3% BSA (Sigma) for 60 min followed by quick washes in TBS-T. Membranes were probed with goat polyclonal anti-PDK2 and PDK4 antibodies according to the manufacturer’s guidelines. The membranes were exposed to chemiluminescent substrate (ECL Advance Western blotting detection kit; GE Health Care, Piscataway, NJ) and imaged using the VersaDoc imaging system (Bio-Rad). Mouse monoclonal antitubulin antibody was used as internal control. Proteins were quantified by densitometric analysis of the blots using Image J software (rsb.info.nih.gov/ij).

Statistical analysis

All assays were performed in triplicate or quadruplicate unless otherwise noted. Data are expressed as mean ± sem of all values obtained in two to five independent experiments. Differences among sample means were tested by ANOVA, followed by the Neuman-Keuls or Bonferroni tests of multiple comparisons. A P < 0.05 was considered statistically significant.

Results

Effect of PRL on basal insulin secretion and GSIS in isolated rat islets

To test the effect of PRL on insulin secretion, we incubated rat islets for 24 h in serum-free basal medium (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm). The islets were washed and preincubated in a secretion buffer containing 2.5 mm glucose. The islets were then transferred to fresh secretion buffer (2.5 mm glucose) and incubated for an additional hour for measurement of basal insulin secretion in conditioned media. The islets were then transferred to fresh secretion buffer containing 16.7 mm glucose. After an incubation lasting 1 h, the conditioned media were collected for measurement of GSIS. As shown in Fig. 1, prior treatment with PRL for 24 h had no significant effect on basal insulin concentrations normalized per microgram protein. After incubation with high glucose (16.7 mm), however, insulin levels in the conditioned media were 37% higher in PRL-treated cells than diluent-treated cells (n = 8, P < 0.001). Similar effects were observed in our previous studies using rat insulinoma (832–13) cells (18): PRL stimulated a 48% increase in GSIS and reversed the inhibitory effect of DEX.

Figure 1.

Figure 1

Open in a new tab

Effect of PRL on basal insulin secretion and GSIS in isolated rat islets. Isolated rat islets were incubated in RPMI 1640 containing 10% FCS and 6.8 mm glucose (growth medium). After 48 h in growth medium, the islets were washed and incubated in serum-free RPMI (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm) for 24 h. Insulin secretion was measured as described in Materials and Methods. The figures show the means ± sem of all data from two independent experiments, each of which contained four samples per treatment group. ***, Statistically significant differences between the experimental groups (P < 0.001).

Effect of glucose, PRL, and DEX on PC mRNA levels

Several investigators have provided evidence that mitochondrial metabolism is coupled to insulin secretion through control of pyruvate metabolism by PC (35,36,37,38,39); GSIS correlates strongly with PC-catalyzed anaplerotic flux into the trichloroacetic acid cycle and stimulation of pyruvate cycling activity. We therefore examined the effects of PRL, glucose, and DEX on PC expression in isolated rat islets and INS-1 cells.

PC is regulated at least in part at the level of transcription. Our initial studies examined the levels of PC mRNA in islets and INS-1 cells incubated at varying glucose concentrations. mRNA levels were analyzed after a 24-h incubation in serum-free medium containing 2.5, 5.5, or 11 mm glucose. As shown in Fig. 2, the levels of PC mRNA in islets incubated in 11 mm glucose were 63.7% (P < 0.001) higher than in islets incubated in 2.5 or 5.5 mm glucose. Glucose had no significant effect on PC mRNA levels in INS-1 cells. PRL reduced the levels of PC mRNA by 41–55% (P < 0.001) in islets incubated in 5.5–11 mm glucose and by 20–36% (P < 0.05–0.01) in INS-1 cells. PRL also reduced PC mRNA in islets and INS-1 cells incubated in DEX, which alone had minimal or no significant effect on PC mRNA (Fig. 3). Neither PRL nor DEX had a statistically significant effect on PC protein, as assessed by Western blot (data not shown), or on PC activity, as assessed by [14C] radioactivity incorporated into dicarboxylic acids (islets: control, 22.5 ± 2.3 vs. PRL 20.06 ± 4.18 nmol/mg protein · h; INS-1 cells: control, 5.85 ± 0.98, PRL, 8.06 ± 0.69, DEX, 7.23 ± 0.49, PRL + DEX, 4.18 ± 0.87 nmol/mg protein · h).

Figure 2.

Figure 2

Open in a new tab

Effects of hormones and varying glucose concentrations on PC mRNA levels in islets and INS-1 cells. A, Islets were incubated for 24 h in serum-free RPMI 1640 containing varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA) in the presence or absence of rat PRL. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control CT value was 25 (Table 1). Values represent the means ± sem of four samples in a representative experiment. ***, Statistically significant differences between the experimental groups (P < 0.001). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.1% HSA) in the presence or absence of PRL. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments.

Figure 3.

Figure 3

Open in a new tab

Effects of PRL and DEX on PC mRNA expression in rat islets and INS-1 cells. A, PC mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two (PRL + DEX). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media (5.5 mm glucose + 0.1% HSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments.

Effect of PRL and glucose on β-cell PDH activity

Insulin secretion correlates with the flux of glucose through PDH well as PC. In contrast to PC, PDH activity is regulated at the level of phosphorylation rather than transcription; PDH is activated by dephosphorylation and inactivated by phosphorylation. There are no commercially available antibodies to measure the phosphorylation state of PDH by Western blot; however, activation of PDH can be assessed by measuring the liberation of 14CO2 from [1-14C] pyruvate.

In preliminary studies, we found that measurements of PDH activity in isolated islets were impossible to interpret; nonspecific counts equaled or exceeded the calculated levels of PDH activity, possibly because total cellular protein content was low. We therefore examined the effects of PRL and glucose on PDH enzyme activity in INS-1 cells; the higher concentrations of protein in INS-1 cell extracts permitted reliable assessments of PDH activity.

As shown in Fig. 4A, an increase in ambient glucose concentrations stimulated an increase (P < 0.01) in PDH activity in INS-1 cells (2.5 mm, 0.43 ± 0.05 nmol/mg protein · h; 5.5 mm, 0.98 ± 0.10 nmol/mg protein · h; 11 mm, 1.94 ± 0.27 nmol/mg protein · h). PRL increased PDH activity in INS-1 cells (+35–63%, P < 0.05–0.001) at all glucose concentrations. The combination of 11 mm glucose plus PRL appeared to have an additive effect; PDH activity in cells incubated in 11 mm glucose plus PRL was 63% higher (P < 0.05) than in INS-1 cells incubated in 2.5 mm glucose plus PRL.

Figure 4.

Figure 4

Open in a new tab

Effects of PRL, glucose, and DEX on β-cell PDH activity. A, INS-1 cells were grown in RPMI 1640 (11.1 mm glucose) supplemented with 10% FBS (growth medium); at 80% confluence the cells were washed and incubated for 24 h in serum-free basal medium in the presence or absence of PRL (20 nm) at varying glucose concentrations (2.5, 5.5, and 11 mm). PDH enzyme activity was estimated by measuring β-cell radioactive CO2 production from [1-14CO2] pyruvate. Values represent the means ± sem of six samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in three independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal medium in the presence or absence of rat PRL (20 nm), DEX (100 nm), or a combination of the two. Values represent the means ± sem of six samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in three independent experiments.

We next examined the interactions of PRL with DEX, a glucocorticoid that inhibits GSIS in isolated islets and insulinoma cells (18,20,22,40,41,42). In INS-1 cells incubated in 5.5 mm glucose, DEX (100 nm) reduced PDH activity by 90% (P < 0.001, Fig. 4B). In contrast, PRL (20 nm) stimulated a 2-fold increase (P < 0.01) in PDH activity and reversed the effects of DEX (P < 0.05).

Expression of PDK mRNA isoforms in insulinoma cells and islets

PDH is activated by PDH phosphatase and inactivated by PDKs (43). Long-term modulation of PDH is mediated in part by transcriptional regulation of PDK genes (43). In the liver, PDK expression is increased by starvation, diabetes, and glucocorticoids and reduced by insulin (44,45,46,47,48). We therefore characterized the expression of PDK mRNAs in rat islets and insulinoma cells and examined their regulation by PRL, glucose, and DEX.

The PDKs are expressed as four isoforms (PDKs 1–4). We used Q-RTPCR to estimate the relative abundance of the various mRNAs in isolated rat islets and INS-1 cells. The mean CT values provide an estimate of the relative abundance of the various mRNAs; the CT value is inversely proportional to the amount of mRNA (Table 1). PDK1, -2, -3, and -4 were expressed in islets as well as INS-1 cells (Table 1), but the levels of PDK4 mRNA in INS-1 cells were about 30,000-fold lower than those in rat islets.

Effects of PRL and glucose on PDK gene expression in islets and INS-1 cells

Our initial studies showed that PRL had no consistent effects on PDK1 or -3 mRNAs in isolated rat islets or INS-1 cells. However, PRL reduced the levels of PDK2 and -4 mRNAs in islets and PDK2 mRNA in INS-1 cells. We therefore examined the interactions of PRL, glucose, and DEX in the regulation of PDK2 and PDK4 expression.

As shown in Fig. 5, A and B, PDK2 mRNA levels were 2-fold higher (P < 0.01) in islets and 22% higher (P < 0.01) in INS-1 cells incubated in 11 mm glucose than in islets or INS-1 cells incubated in 2.5 or 5.5 mm glucose. PRL reduced by 50% (P < 0.01) the expression of PDK2 mRNA in islets incubated in 5.5 mm glucose and by 25–42% (P < 0.05–0.001) the expression of PDK2 mRNA in INS-1 cells. DEX increased PDK2 mRNA in islets and INS-1 cells; this effect was reversed by coincubation with PRL (Fig. 6, A and B).

Figure 5.

Figure 5

Open in a new tab

Effects of PRL and glucose on PDK2 mRNA levels in islets and INS-1 cells. A, Islets were incubated for 24 h in serum-free RPMI 1640 with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control CT value was 25 (Table 1). Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.1% HSA) in the presence or absence of PRL. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in four independent experiments.

Figure 6.

Figure 6

Open in a new tab

Effects of PRL and DEX on PDK2 expression in rat islets and INS-1 cells. A, PDK2 mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Similar results were obtained in four independent experiments. B, INS-1 cells were incubated for 24 h in serum-free basal media (5.5 mm glucose + 0.1% HSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (***, P < 0.001). Similar results were obtained in four independent experiments. C, Effects of PRL and DEX on PDK2 protein levels in rat islets. Rat islets were incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of rat PRL (20 nm), DEX (100 nm), or a combination of the two. Protein expression was analyzed by Western blot. The values were corrected for γ-tubulin. Similar results were obtained in three independent experiments.

PDK2 protein levels were assessed by Western blot (Fig. 6C). PRL decreased PDK2 protein levels by 34.3% (P < 0.05) compared with control. DEX alone had no significant effect on PDK2 protein levels. The levels of PDK2 protein were 66.4% (P < 0.01) lower in islets treated with PRL + DEX than islets treated with DEX alone.

As shown in Fig. 7A, PDK4 mRNA levels were 2-fold higher (P < 0.001) in islets incubated in 11 mm glucose than islets incubated in 2.5 or 5.5 mm glucose. PRL reduced PDK4 mRNA by 30–80% (P < 0.001) in islets incubated at 5.5 or 11 mm glucose. DEX had no significant effect on PDK4 mRNA but attenuated the effect of PRL (Fig. 7B). Attempts to quantify PDK4 protein by Western blot were unsuccessful because of high nonspecific binding of antisera to cell proteins fractionated by electrophoresis.

Figure 7.

Figure 7

Open in a new tab

Effects of PRL and glucose on PDK4 mRNA levels in rat islets. A, Islets were incubated for 24 h in serum-free RPMI 1640 with varying glucose concentrations (2.5, 5.5, and 11 mm, 0.5% BSA). mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. The control CT value was 21 (Table 1). Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments. B, PDK4 mRNA levels were measured in islets incubated for 24 h in serum-free media (5.5 mm glucose, 0.5% BSA) in the presence or absence of PRL (20 nm), DEX (100 nm), or a combination of the two. mRNA levels were measured by Q-RTPCR. Values in diluent-treated control cells were adjusted so that the mean equaled 1.0; values for hormone-treated cells were calculated as a function of the mean of control values. Values represent the means ± sem of four samples in a representative experiment. Statistically significant differences between the experimental groups are indicated (*, P < 0.05; ***, P < 0.001). Similar results were obtained in four independent experiments.

Discussion

The mechanisms by which PRL regulates β-cell insulin production have not been elucidated. PRL increases glucose transport, glucose transporter 2 mRNA, and glucokinase activity in islets and insulinoma cells (13,14,20,22,49,50), suggesting that its induction of GSIS is mediated in part by glucose uptake and utilization. A major step in glucose utilization is the glycolytic generation of pyruvate. In the current study, we explored the interactions of PRL, glucose, and glucocorticoids in the regulation of PC and PDH, which in pancreatic β-cells contribute equally to the flux of pyruvate into the Krebs cycle (51,52).

Whereas glucose at supraphysiologic concentrations (11 mm) stimulated an increase in PC mRNA in rat islets, PRL reduced PC mRNA in islets and INS-1 cells. The inhibitory effect of PRL was noted in the presence or absence of DEX. However, PRL had no effect on PC protein levels, assessed by Western blot, or PC activity, assessed by [14C] radioactivity incorporated into dicarboxylic acids. Thus, the induction of GSIS by PRL cannot be explained by induction of PC expression. Rather, PRL induction of GSIS correlates with induction of PDH activity. Like glucose, PRL stimulated an increase in PDH activity in INS-1 cells and reversed the effect of DEX, which inhibited PDH. The magnitude of the PRL effect on PDH was greater at 2.5 and 5.5 mm than at 11 mm glucose, suggesting that PRL action is mediated through mechanisms distinct in part from glucose uptake or utilization.

PDH is activated by PDH phosphatase and inactivated by PDKs (43). We showed that PDKs 1–4 are expressed in rat islets. In contrast, INS-1 cells express PDKs 1–3 but little or no PDK4. Supraphysiological concentrations of glucose (11 mm) induced expression of PDK2 and -4 mRNAs in islets and INS-1 cells; in contrast, PRL decreased the levels of PDK2 mRNA and protein in islets and INS-1 cells and reduced PDK4 mRNA in rat islets. PRL also reversed the induction of PDK2 by DEX. These findings suggest that the opposing effects of PRL and DEX on GSIS are mediated in part through opposing effects on PDK-regulated PDH activity. Given that 11 mm glucose stimulated increases in PDK2 and PDK4 mRNAs in islets and INS-1 cells, the effects of PRL on PDK expression appear to be distinct from, or to overlap with, the hormone’s effects on glucose transport or use.

The mechanisms by which PRL and DEX regulate PDK expression are currently unclear. One possible pathway involves the regulation of PDK mRNA by the forkhead protein FoxO1. FoxO1 binds to the PDK4 promoter and up-regulates PDK4 expression (43,53). In recent studies we demonstrated that PRL down-regulates and DEX up-regulates nuclear expression of FoxO1 in β-cells (18).

Fasting, malnutrition, and diabetes are associated with increases in hepatic PDK expression and reductions in hepatic and β-cell PDH activity (44,54,55). Thus, suppression of PDK and up-regulation of PDH in β-cells may counteract the effects of nutrient deprivation, diabetes, and metabolic stress. Interestingly, overexpression of placental lactogen in β-cells in vivo protects against the development of diabetes in streptozotocin-treated mice (10,56,57). In recent manuscripts (18,49,58), we posited that the lactogenic hormones may maintain β-cell mass and function under conditions of stress and nutrient deprivation and may thereby protect against the development of gestational or glucocorticoid-dependent diabetes mellitus. Our demonstration that PRL reverses the effects of DEX on PDK expression and PDH activity is consistent with this hypothesis.

Acknowledgments

We thank Drs. Mette V. Jensen and Sam Stephens (Sarah W. Stedman Nutrition Center) for providing suggestions for some experiments included in this paper. We also thank the National Hormone and Peptide Program and Dr. A. F. Parlow, scientific director (Torrance, CA) for providing rat PRL.

Footnotes

This work was supported by Grant HD024192 from the National Institutes of Health, National Institute of Child Health and Human Development, Grant 7-08-RA-46 from the American Diabetes Association, and grants from the Pfizer Corp. (to M.F.) and the Duke Children’s Miracle Network (to R.A.).

Disclosure Summary: The authors have nothing to declare.

First Published Online May 19, 2010

Abbreviations: BCA, Bicinchoninic acid; CT, threshold cycle; DEX, dexamethasone; FBS, fetal bovine serum; FoxO, Forkhead box O; GSIS, glucose-stimulated insulin secretion; HSA, human serum albumin; INS-1, insulinoma; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PL, placental lactogen; PRL, prolactin; Q-RTPCR, quantitative real-time PCR; RIPA, radioimmunoprecipitation assay; TBS-T, Tris-buffered and Tween 20.

References

  1. Nielsen JH 1982 Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets. Endocrinology 110:600–606 [DOI] [PubMed] [Google Scholar]
  2. Swenne I, Hill DJ, Strain AJ, Milner RD 1987 Effects of human placental lactogen and growth hormone on the production of insulin and somatomedin C/insulin-like growth factor I by human fetal pancreas in tissue culture. J Endocrinol 113:297–303 [DOI] [PubMed] [Google Scholar]
  3. Brelje TC, Sorenson RL 1991 Role of prolactin versus growth hormone on islet B cell proliferation in vitro: implications for pregnancy. Endocrinology 128:45–57 [DOI] [PubMed] [Google Scholar]
  4. Brelje TC, Parsons JA, Sorenson RL 1994 Regulation of islet β-cell proliferation by prolactin in rat islets. Diabetes 43:263–273 [DOI] [PubMed] [Google Scholar]
  5. Møldrup A, Petersen ED, Nielsen JH 1993 Effects of sex and pregnancy hormones on growth hormone and prolactin receptor gene expression in insulin-producing cells. Endocrinology 133:1165– 1172 [DOI] [PubMed] [Google Scholar]
  6. Freemark M, Nagano M, Edery M, Kelly PA 1995 Prolactin receptor gene expression in the fetal rat. J Endocrinol 144:285–292 [DOI] [PubMed] [Google Scholar]
  7. Royster M, Driscoll P, Kelly PA, Freemark M 1995 The prolactin receptor in the fetal rat: cellular localization of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation. Endocrinology 136:3892–3900 [DOI] [PubMed] [Google Scholar]
  8. Freemark M, Driscoll P, Maaskant R, Petryk A, Kelly PA 1997 Ontogenesis of prolactin receptors in the human fetus in early gestation. Implications for tissue differentiation and development. J Clin Invest 99:1107–1117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fleenor D, Petryk A, Driscoll P, Freemark M 2000 Constitutive expression of placental lactogen in pancreatic β cells: effects on cell morphology, growth, and gene expression. Pediatr Res 47:136–142 [DOI] [PubMed] [Google Scholar]
  10. Vasavada RC, Garcia-Ocaña A, Zawalich WS, Sorenson RL, Dann P, Syed M, Ogren L, Talamantes F, Stewart AF 2000 Targeted expression of placental lactogen in the β cells of transgenic mice results in β cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem 275:15399–15406 [DOI] [PubMed] [Google Scholar]
  11. Freemark M, Avril I, Fleenor D, Driscoll P, Petro A, Opara E, Kendall W, Oden J, Bridges S, Binart N, Breant B, Kelly PA 2002 Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology 143: 1378–1385 [DOI] [PubMed] [Google Scholar]
  12. Huang C, Snider F, Cross JC 2009 Prolactin receptor is required for normal glucose homeostasis and modulation of β-cell mass during pregnancy. Endocrinology 150:1618–1626 [DOI] [PubMed] [Google Scholar]
  13. Petryk A, Fleenor D, Driscoll P, Freemark M 2000 Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2. J Endocrinol 164:277–286 [DOI] [PubMed] [Google Scholar]
  14. Weinhaus AJ, Stout LE, Bhagroo NV, Brelje TC, Sorenson RL 2007 Regulation of glucokinase in pancreatic islets by prolactin: a mechanism for increasing glucose-stimulated insulin secretion during pregnancy. J Endocrinol 193:367–381 [DOI] [PubMed] [Google Scholar]
  15. Rubí B, Antinozzi PA, Herrero L, Ishihara H, Asins G, Serra D, Wollheim CB, Maechler P, Hegardt FG 2002 Adenovirus-mediated overexpression of liver carnitine palmitoyltransferase I in INS1E cells: effects on cell metabolism and insulin secretion. Biochem J 364:219–226 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Herrero L, Rubí B, Sebastián D, Serra D, Asins G, Maechler P, Prentki M, Hegardt FG 2005 Alteration of the malonyl-CoA/carnitine palmitoyltransferase I interaction in the β-cell impairs glucose-induced insulin secretion. Diabetes 54:462–471 [DOI] [PubMed] [Google Scholar]
  17. Martinez SC, Cras-Méneur C, Bernal-Mizrachi E, Permutt MA 2006 Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet β-cell. Diabetes 55:1581–1591 [DOI] [PubMed] [Google Scholar]
  18. Arumugam R, Horowitz E, Lu D, Collier JJ, Ronnebaum S, Fleenor D, Freemark M 2008 The interplay of prolactin and the glucocorticoids in the regulation of β-cell gene expression, fatty acid oxidation, and glucose-stimulated insulin secretion: implications for carbohydrate metabolism in pregnancy. Endocrinology 149:5401–5414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Aerts L, Assche FA 1975 Ultrastructural changes of the endocrine pancreas in pregnant rats. Diabetologia 11:285–289 [DOI] [PubMed] [Google Scholar]
  20. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL 2000 Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology 141:1384–1393 [DOI] [PubMed] [Google Scholar]
  21. Fleenor DE, Freemark M 2001 Prolactin induction of insulin gene transcription: roles of glucose and signal transducer and activator of transcription 5. Endocrinology 142:2805–2810 [DOI] [PubMed] [Google Scholar]
  22. Shao J, Qiao L, Friedman JE 2004 Prolactin, progesterone, and dexamethasone coordinately and adversely regulate glucokinase and cAMP/PDE cascades in MIN6 β-cells. Am J Physiol Endocrinol Metab 286:E304–E310 [DOI] [PubMed] [Google Scholar]
  23. Hügl SR, Merger M 2007 Prolactin stimulates proliferation of the glucose-dependent β-cell line INS-1 via different IRS-proteins. J Pancreas 8:739–752 [PubMed] [Google Scholar]
  24. Milburn Jr JL, Hirose H, Lee YH, Nagasawa Y, Ogawa A, Ohneda M, BeltrandelRio H, Newgard CB, Johnson JH, Unger RH 1995 Pancreatic β-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem 270:1295–1299 [DOI] [PubMed] [Google Scholar]
  25. Freemark M 1999 The roles of growth hormone, prolactin and placental lactogen in human fetal development. Critical analysis of molecular, cellular and clinical investigations. In: Handwerger S, ed. Molecular and cellular pediatric endocrinology. Totowa, NJ: Humana; 57–83 [Google Scholar]
  26. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 [DOI] [PubMed] [Google Scholar]
  27. Dheda K Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A 2004 Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37:112–114, 118–119 [DOI] [PubMed] [Google Scholar]
  28. Jensen MV, Joseph JW, Ilkayeva O, Burgess S, Lu D, Ronnebaum SM, Odegaard M, Becker TC, Sherry AD, Newgard CB 2006 Compensatory responses to pyruvate carboxylase suppression in islet β-cells. Preservation of glucose-stimulated insulin secretion. J Biol Chem 281:22342–22351 [DOI] [PubMed] [Google Scholar]
  29. Kim JY, Hickner RC, Cortright RL, Dohm GL, Houmard JA 2000 Lipid oxidation is reduced in obese human skeletal muscle. Am J Physiol Endocrinol Metab 279:E1039–E1044 [DOI] [PubMed] [Google Scholar]
  30. Kim JY, Koves TR, Yu GS, Gulick T, Cortright RN, Dohm GL, Muoio DM 2002 Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle. Am J Physiol Endocrinol Metab 282:E1014–E1022 [DOI] [PubMed] [Google Scholar]
  31. MacDonald MJ 1995 Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion. J Biol Chem 270:20051–20058 [PubMed] [Google Scholar]
  32. MacDonald MJ, Tang J, Polonsky KS 1996 Low mitochondrial glycerol phosphate dehydrogenase and pyruvate carboxylase in pancreatic islets of Zucker diabetic fatty rats. Diabetes 45:1626–1630 [DOI] [PubMed] [Google Scholar]
  33. Abdel-aleem S, Sayed-Ahmed M, Nada MA, Hendrickson SC, St. Louis J, Lowe JE 1995 Stimulation of non-oxidative glucose utilization by l-carnitine in isolated myocytes. J Mol Cell Cardiol 27:2465–2472 [DOI] [PubMed] [Google Scholar]
  34. Cortright RN, Sandhoff KM, Basilio JL, Berggren JR, Hickner RC, Hulver MW, Dohm GL, Houmard JA 2006 Skeletal muscle fat oxidation is increased in African-American and white women after 10 days of endurance exercise training. Obesity (Silver Spring) 14:1201–1210 [DOI] [PubMed] [Google Scholar]
  35. Corkey BE, Glennon MC, Chen KS, Deeney JT, Matschinsky FM, Prentki M 1989 A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic β-cells. J Biol Chem 264:21608–216012 [PubMed] [Google Scholar]
  36. Prentki M 1996 New insights into pancreatic β-cell metabolic signaling in insulin secretion. Eur J Endocrinol 134:272–286 [DOI] [PubMed] [Google Scholar]
  37. Prentki M, Tornheim K, Corkey BE 1997 Signal transduction mechanisms in nutrient-induced insulin secretion. Diabetologia 40(Suppl 2):S32–S41 [DOI] [PubMed] [Google Scholar]
  38. Deeney JT, Gromada J, Høy M, Olsen HL, Rhodes CJ, Prentki M, Berggren PO, Corkey BE 2000 Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI β-cells). J Biol Chem 275:9363–9368 [DOI] [PubMed] [Google Scholar]
  39. Farfari S, Schulz V, Corkey B, Prentki M 2000 Glucose-regulated anaplerosis and cataplerosis in pancreatic β-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion. Diabetes 49:718–726 [DOI] [PubMed] [Google Scholar]
  40. Lambillotte C, Gilon P, Henquin JC 1997 Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. J Clin Invest 99:414–423 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ullrich S, Berchtold S, Ranta F, Seebohm G, Henke G, Lupescu A, Mack AF, Chao CM, Su J, Nitschke R, Alexander D, Friedrich B, Wulff P, Kuhl D, Lang F 2005 Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates glucocorticoid-induced inhibition of insulin secretion. Diabetes 54:1090–1099 [DOI] [PubMed] [Google Scholar]
  42. Ranta F, Avram D, Berchtold S, Düfer M, Drews G, Lang F, Ullrich S 2006 Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes 55:1380–1390 [DOI] [PubMed] [Google Scholar]
  43. Xu J, Han J, Epstein PN, Liu YQ 2006 Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets. Biochem Biophys Res Commun 344:827–833 [DOI] [PubMed] [Google Scholar]
  44. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM 1998 Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 329(Pt 1):191–196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sugden MC, Fryer LG, Orfali KA, Priestman DA, Donald E, Holness MJ 1998 Studies of the long-term regulation of hepatic pyruvate dehydrogenase kinase. Biochem J 329(Pt 1):89–94 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sugden MC, Holness MJ, Donald E, Lall H 1999 Substrate interactions in the short- and long-term regulation of renal glucose oxidation. Metabolism 48:707–715 [DOI] [PubMed] [Google Scholar]
  47. Wu P, Blair PV, Sato J, Jaskiewicz J, Popov KM, Harris RA 2000 Starvation increases the amount of pyruvate dehydrogenase kinase in several mammalian tissues. Arch Biochem Biophys 381:1–7 [DOI] [PubMed] [Google Scholar]
  48. Sugden MC, Bulmer K, Gibbons GF, Knight BL, Holness MJ 2002 Peroxisome-proliferator-activated receptor-α (PPARα) deficiency leads to dysregulation of hepatic lipid and carbohydrate metabolism by fatty acids and insulin. Biochem J 364:361–368 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Freemark M 2006 Regulation of maternal metabolism by pituitary and placental hormones: roles in fetal development and metabolic programming. Horm Res 65(Suppl 3):S41–S49 [DOI] [PubMed] [Google Scholar]
  50. Sorenson RL, Brelje TC 2009 Prolactin receptors are critical to the adaptation of islets to pregnancy. Endocrinology 150:1566–1569 [DOI] [PubMed] [Google Scholar]
  51. Khan A, Ling ZC, Landau BR 1996 Quantifying the carboxylation of pyruvate in pancreatic islets. J Biol Chem 271:2539–2542 [DOI] [PubMed] [Google Scholar]
  52. Xu J, Han J, Long YS, Epstein PN, Liu YQ 2008 The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic β cells. Diabetologia 51:2022–2030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Furuyama T, Kitayama K, Yamashita H, Mori N 2003 Forkhead transcription factor FOXO1 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation. Biochem J 375:365–371 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhou YP, Ostenson CG, Ling ZC, Grill V 1995 Deficiency of pyruvate dehydrogenase activity in pancreatic islets of diabetic GK rats. Endocrinology 136:3546–3551 [DOI] [PubMed] [Google Scholar]
  55. Patel MS, Korotchkina LG 2001 Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. Exp Mol Med 33:191–197 [DOI] [PubMed] [Google Scholar]
  56. Fujinaka Y, Sipula D, Garcia-Ocaña A, Vasavada RC 2004 Characterization of mice doubly transgenic for parathyroid hormone-related protein and murine placental lactogen: a novel role for placental lactogen in pancreatic β-cell survival. Diabetes 53:3120–3130 [DOI] [PubMed] [Google Scholar]
  57. Fujinaka Y, Takane K, Yamashita H, Vasavada RC 2007 Lactogens promote β cell survival through JAK2/STAT5 activation and Bcl-XL upregulation. J Biol Chem 282:30707–30717 [DOI] [PubMed] [Google Scholar]
  58. Arumugam R, Fleenor D, Freemark M 2007 Effects of lactogen resistance and GH deficiency on mouse metabolism: pancreatic hormones, adipocytokines, and expression of adiponectin and insulin receptors. Endocrine 32:182–191 [DOI] [PubMed] [Google Scholar]

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES