5′AMP-activated protein kinase activity is increased in adipose tissue of northern elephant seal pups during prolonged fasting-induced insulin resistance

in Journal of Endocrinology

Northern elephant seals endure a 2- to 3-month fast characterized by sustained hyperglycemia, hypoinsulinemia, and increased plasma cortisol and free fatty acids, conditions often seen in insulin-resistant humans. We had previously shown that adipose Glut4 expression and 5′AMP-activated protein kinase (AMPK) activity increase and plasma glucose decreases in fasting seals suggesting that AMPK activity contributes to glucose regulation during insulin-resistant conditions. To address the hypothesis that AMPK activity increases during fasting-induced insulin resistance, we performed glucose tolerance tests (GTT) on early (n=5) and late (n=8)-fasted seal pups and compared adipose tissue expression of insulin signaling proteins, peroxisome proliferator-activated receptor γ (PPARγ), and AMPK, in addition to plasma adiponectin, leptin, cortisol, insulin, and non-esterified fatty acid (NEFA) levels. Fasting was associated with decreased glucose clearance, plasma insulin and adiponectin, and intracellular insulin signaling, as well as increased plasma cortisol and NEFAs, supporting the suggestion that seals develop insulin resistance late in the fast. The expression of Glut4 and VAMP2 increased (52 and 63% respectively) with fasting but did not change significantly during the GTT. PPARγ and phosphorylated AMPK did not change in the early fasted seals, but increased significantly (73 and 50% respectively) in the late-fasted seals during the GTT. Increased AMPK activity along with the reduction in the activity of insulin-signaling proteins supports our hypothesis that AMPK activity is increased following the onset of insulin resistance. The association between increased AMPK activity and Glut4 expression suggests that AMPK plays a greater role in regulating glucose metabolism in mammals adapted to prolonged fasting than in non-fasting mammals.

Abstract

Northern elephant seals endure a 2- to 3-month fast characterized by sustained hyperglycemia, hypoinsulinemia, and increased plasma cortisol and free fatty acids, conditions often seen in insulin-resistant humans. We had previously shown that adipose Glut4 expression and 5′AMP-activated protein kinase (AMPK) activity increase and plasma glucose decreases in fasting seals suggesting that AMPK activity contributes to glucose regulation during insulin-resistant conditions. To address the hypothesis that AMPK activity increases during fasting-induced insulin resistance, we performed glucose tolerance tests (GTT) on early (n=5) and late (n=8)-fasted seal pups and compared adipose tissue expression of insulin signaling proteins, peroxisome proliferator-activated receptor γ (PPARγ), and AMPK, in addition to plasma adiponectin, leptin, cortisol, insulin, and non-esterified fatty acid (NEFA) levels. Fasting was associated with decreased glucose clearance, plasma insulin and adiponectin, and intracellular insulin signaling, as well as increased plasma cortisol and NEFAs, supporting the suggestion that seals develop insulin resistance late in the fast. The expression of Glut4 and VAMP2 increased (52 and 63% respectively) with fasting but did not change significantly during the GTT. PPARγ and phosphorylated AMPK did not change in the early fasted seals, but increased significantly (73 and 50% respectively) in the late-fasted seals during the GTT. Increased AMPK activity along with the reduction in the activity of insulin-signaling proteins supports our hypothesis that AMPK activity is increased following the onset of insulin resistance. The association between increased AMPK activity and Glut4 expression suggests that AMPK plays a greater role in regulating glucose metabolism in mammals adapted to prolonged fasting than in non-fasting mammals.

Keywords:

Introduction

Many factors contribute to the development of insulin resistance including impaired hormone secretion and/or activity, reductions in the expression of key signaling proteins, and reduced protein activity through inhibition (Mothe & van Obberghen 1996, Perseghin et al. 2003, Cornier et al. 2006). As insulin regulates numerous critical physiological functions, such as cellular glucose uptake, the pancreas compensates for diminished insulin sensitivity by secreting more insulin (Czech 1995, Leney & Tavare 2009). Once the pancreas reaches a point where it can no longer produce sufficient insulin to elicit a cellular effect in insulin-responsive tissues, glucose clearance becomes impaired and persistent hyperglycemia occurs (i.e. type 2 diabetes mellitus) (Campbell 2009, Lin & Sun 2010). Various hormones and proteins, such as adiponectin, glucagon-like peptide-1 (GLP-1), insulin-like growth factor 1 (IGF1), and peroxisome proliferator-activated receptor γ (PPARγ), are insulinotropic and facilitate insulin sensitivity and function in peripheral tissues (Ahima & Lazar 2008, Arafat et al. 2010, Cefalu 2010, Hsiao et al. 2011). However, the circulating concentrations of these factors, or their intracellular expression, are usually decreased in diabetic patients (Gregoire et al. 1998, Kahn & Flier 2000, Knop et al. 2007, Kouidhi et al. 2010). At the same time, cortisol, which inhibits insulin secretion from the pancreas and stimulates hepatic gluconeogenesis (the generation of glucose from non-carbohydrate substrates), and free fatty acids, which decrease insulin sensitivity by inhibiting intracellular insulin signaling and glucose uptake, are increased in the circulation, contributing to continuation of the diabetic state (Khan et al. 1992, Epps-Fung et al. 1997, Khani & Tayek 2001, Lam et al. 2003, Macfarlane et al. 2008, van Raalte 2009).

Similar conditions are often observed in mammals chronically deprived of food, including humans, as stress-induced increases in plasma cortisol, paired with the mobilization of limited metabolic substrates, lead to dysregulation of carbohydrate and lipid metabolism (Saboureau et al. 1980, Scheen et al. 1988, Krassas 2003). This results in reductions in the concentrations of insulinotropic hormones and eventually leads to loss of sensitivity to insulin (Scheen et al. 1988, Krassas 2003). Although prolonged fasting-induced insulin resistance may be detrimental to most mammals, elephant seals have evolved robust physiological mechanisms that have allowed them to adapt to a fasting lifestyle and endure months without food or exogenous water.

The prolonged, fast of the elephant seal is characterized by hyperglycemia, along with increasing plasma cortisol and free fatty acids (Costa & Ortiz 1982, Castellini et al. 1987, Keith & Ortiz 1988, Ortiz et al. 2001, Champagne et al. 2005). Insulin signaling, plasma adiponectin, and the expression of adipose tissue PPARγ decrease during the fasting of elephant seals, suggesting that the seal becomes insulin resistant late in the fast (Viscarra et al. 2011). However, expression of Glut4 and phosphorylation of 5′AMP-activated protein kinase (AMPK) in adipose are increased (Viscarra et al. 2011). Increased AMPK activity can stimulate Glut4 translocation (Russell et al. 1999), suggesting that glucose uptake is not compromised late in the seal's fast. Apart from the contributions that can be made to our understanding of the regulation of metabolic substrates during prolonged fasting, because elephant seals do not appear to be as susceptible to insulin resistance as humans (the health detriments associated with insulin resistance have not been observed in the seal (Castellini & Costa 1990, Adams & Costa 1993, Champagne et al. 2005)), the elephant seal provides a unique opportunity to promote and enhance our knowledge of the effects of, as well as the adaptation to, insulin resistance.

The goal of this study was to examine the intracellular changes associated with insulin signaling and carbohydrate metabolism that occur as a result of the hormonal and biochemical flux that elephant seals experience during prolonged fasting. We had previously shown that AMPK activity is increased in late-fasted elephant seals, suggesting that it may contribute to increased Glut4 translocation. To examine our hypothesis that AMPK activity in adipose tissue increases during reduced insulin signaling, we performed a glucose tolerance test (GTT) on early- and late-fasted elephant seals. The GTT allowed us to 1) gauge the sensitivity of the pancreas and adipose to glucose and 2) to examine the response of the insulin signaling pathway and facilitating proteins in adipose tissue to better ascertain the mechanisms induced following the onset of insulin resistance.

Materials and Methods

All procedures were reviewed and approved by the Institutional Animal Care and Use Committee's of both the University of California Merced and the Sonoma State University. All work was conducted under the National Marine Fisheries Service marine mammal permit #87-1743.

GTT and sample collection

In total, 13 northern elephant seal (Mirounga angustirostris) pups were studied at the Año Nuevo State Reserve (30 km north of Santa Cruz, CA, USA) during their natural post-weaning fast while they are still on land. The pups were sampled at two time periods: early (2–3 week post-weaning (Feb 2010); n=5) and late (6–8 week post-weaning (March 2010); n=8) fasting. The elephant seal pups were isolated during the procedures to avoid interruption by the much larger adults. The body mass was measured using a hanging-load cell suspended from a tripod. After weighing, the pups were initially sedated with 1 mg/kg Telazol (tiletamine/zolazepam HCl, Fort Dodge Labs, Ft Dodge, IA, USA) administrated intramuscularly. Once immobilized, an 18 gauge, 3.5-inch spinal needle was inserted into the extradural spinal vein. Blood glucose was measured using a commercially available blood glucose monitor (OneTouch Ultra2; LifeScan, Inc., Milpitas, CA, USA). Pre-infusion blood samples were collected in chilled, EDTA-treated vacutainer sample tubes containing a protease inhibitor cocktail (PIC; Sigma–Aldrich) and kept on ice until they could be centrifuged. A pre-infusion adipose tissue biopsy was collected by first cleaning a small region in the flank of the animal near the hind flipper, with alternating wipes of isopropyl alcohol and betadine, followed by a s.c. injection of 2–3 ml lidocaine (Henry Schein, Melville, NY, USA). A small (<1.5 cm) incision was made using a sterile scalpel, and a blubber biopsy (ca. 100–200 mg) was collected with a sterile biopsy punch needle (Henry Schein). The biopsy samples were rinsed with cold, sterile saline, placed in cryogenic vials, immediately frozen by immersion in liquid nitrogen, and stored at −80 °C until later analyses. Following initial sample collection, animals were infused with a mass-specific dose of glucose (0.5 g/kg) administered through the spinal needle over a 2 min period (Kirby & Ortiz 1994, Fowler et al. 2008). Continued immobilization was maintained with ∼100 mg bolus i.v. injections of ketamine as needed. Subsequent blood samples were collected at 5, 10, 15, 20, 30, 45, 60, 90, 120, and 150 min post-infusion, measured for glucose, and placed on ice until they could be processed. Subsequent adipose biopsies were collected at 60 and 150 min post-infusion and stored as described above.

Sample preparation

As described previously (Viscarra et al. 2011), all blood samples were centrifuged for 15 min at 3000 g at 4 °C, and the plasma was transferred to cryo-vials, frozen by immersion in liquid nitrogen, and immediately stored at −80 °C. Frozen tissue samples were homogenized in 500 μl hypotonic buffer containing a PIC and phosphatase inhibitor cocktail (Halt PIC; Thermo, Waltham, MA, USA). The homogenate was then centrifuged at 16 100 g for 15 min and the aqueous layer was aliquoted into a separate tube. TBS (500 μl) containing 1% v/v Triton X-100, 1% w/v SDS, and 1% v/v PPIC was added to the pellet and sonicated, the resulting suspension was then centrifuged at 16 100 g for 15 min, and the aqueous layer was again transferred to a separate tube. Total protein content in both cytosolic and membrane bound fractions was measured by Bradford assay (Bio-Rad Laboratories).

Western blot

Total protein (30 μg) was resolved in 4–15% Tris–HCl SDS gradient gels. Proteins <100 kDa were electroblotted using the Bio-Rad Trans Blot SD semi-dry cell onto 0.45 μm nitrocellulose membranes. Proteins >100 kDa were electroblotted using the Bio-Rad Mini Protean Transfer apparatus onto 0.45 μm nitrocellulose membrane. The membranes were blocked with 3% BSA in PBS containing 0.05% v/v of Tween 20 (PBS-T) and incubated overnight with primary antibodies (diluted 1:500–1:5000) against p-insulin receptor (IR)-β, p-IRS-1/IRS-1, PI3k, AKT2, Glut4, PPARγ, AMPK/p-AMPK, lamin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), IR-β, p-AKT2, β-actin (Assay Designs, Ann Arbor, MI, USA), and GAPDH (Enzo Life Sciences, Farmingdale, NY, USA). The membranes were washed, incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL, USA), re-washed, and developed using the Immun-Star Western C kit (Bio-Rad). The blots were visualized using a Chemi-Doc XRS system (Bio-Rad) and quantified with Bio-Rad's Quantity One software. In addition to consistently loading the same amount of total protein per well (30 μg), densitometry values were further normalized by correcting the densitometry values of a constitutively expressed protein (β-actin, GAPDH, or lamin).

Plasma analyses

The plasma concentrations of total adiponectin (canine; Linco, St Charles, MO, USA), cortisol (Siemens, Los Angeles, CA, USA), GLP-1 (Millipore, Billerica, MA, USA), IGF1 (DSL, Inc., Webster, TX, USA), insulin (guinea pig anti-porcine; Linco), leptin (guinea pig; Millipore), and non-esterified fatty acids (NEFA; Wako Chemicals, Richmond, VA, USA) were measured with commercially available RIA or enzyme immunoassay kits, previously validated for use with elephant seal plasma (Ortiz et al. 2001, 2003, Champagne et al. 2005, Viscarra et al. 2011). All samples were analyzed in duplicate and run in a single assay with intra-assay, the intra-assay percent coefficients of variability of <10% for all assays.

Statistical analysis

The baseline (or T0) measurements (plasma or tissue protein content) of the early and late groups were used to assess the changes in variables as a function of fasting duration and these means (±s.e.m.) were compared by one-way ANOVA using a Fisher's PLSD post hoc test. The rate of glucose disappearance (K) was calculated assuming the 20 min post-infusion sample represented complete dilution of the injected glucose within the total body pool (Champagne et al. 2005, Fowler et al. 2008). K was then calculated by using the linear regression as the negative slope of glucose concentrations from 20 to 150 min post-infusion (Fowler et al. 2008). Mean (±s.e.m.) area-under-the-curve (AUC) was calculated for each variable measured during the GTTs and used to determine integrated changes between early and late fasting groups. Variables that changed as a result of fasting (i.e. different baseline levels) were then corrected by calculating the percent change from baseline. Mean percent changes were calculated for each time point and compared by repeated measures by ANOVA to identify the changes in response to the GTT using a Fisher's PLSD post hoc test. The changes were considered significantly different at P<0.05. Adipose protein content was normalized by percent change and presented as ratios of either phosphorylated protein to total protein, or protein to constitutively expressed protein (actin, lamin, and GAPDH) and compared by ANOVA to determine the changes in response to the GTT. Statistical analyses were performed with StatView5 software (SAS Institute, Inc., Cary, NC, USA).

Results

Fasting-associated changes

Elephant seal pups were, on average, 32% smaller (P<0.01) in the late fasting period compared with the early fasting period (Table 1). Mean blood glucose and plasma adiponectin, insulin, and IGF1 decreased 35% (P<0.05), 23% (P<0.05), 52% (P<0.001), and 66% (P<0.01) respectively, with fasting (Table 1). Mean plasma cortisol, NEFA, and GLP-1 increased (P<0.01) 80, 82, and 233% respectively, with fasting (Table 1). Mean plasma leptin did not change (Table 1).

Table 1

Morphological and biochemical parameters measured prior to the glucose tolerance tests early and late in the fast

ParameterEarlyLate
Body mass (kg)122±583±7
Blood glucose (mM)9.6±0.77.1±0.5*
NEFA (mM)0.85±0.101.55±0.14
Cortisol (nM)264±35477±49
Adiponectin (ng/ml)386±35296±15*
GLP-1 (pM)21.2±4.070.9±9.0
IGF1 (nM)18.3±3.46.1±1.0
Insulin (μU/ml)2.5±0.31.2±0.1
Leptin (nM)0.19±0.020.19±0.01

*, , and denote significant difference from early fasting at P<0.05, P<0.01, and P<0.001 respectively.

The phosphorylation ratios of IR, IRS-1, and AKT2 decreased 46% (P<0.0001), 39% (P<0.001), and 37% (P<0.001) respectively, with fasting (Table 2). The content of PI3k and PPARγ decreased 21% (P<0.05) and 25% (P<0.001) respectively, with fasting (Table 2). The phosphorylation ratio of p-AMPK increased 38% (P<0.01) with fasting (Table 2). The content of Glut4 and VAMP2 increased (P<0.0001) 60 and 64% respectively, with fasting (Table 2).

Table 2

Ratios of proteins measured in adipose of early- and late-fasted seals prior to the glucose tolerance tests

ProteinsEarlyLate
p-IR:IR0.98±0.030.52±0.04§
p-IRS-1:IRS-11.01±0.050.69±0.01
PI3k:β-actin1.05±0.030.82±0.01*
p-AKT2:AKT20.99±0.010.62±0.05
p-AMPK:AMPK1.01±0.071.33±0.08
PPARγ:lamin0.95±0.030.71±0.03
VAMP2:GAPDH0.99±0.071.63±0.04§
Glut4:GAPDH0.95±0.061.52±0.05§

*, , , and § denote significant difference from early fasting at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively.

Response to GTT

Glucose

The rate of glucose disappearance (indicated by the negative slope of the line fitted to glucose concentrations) following the GTTs decreased (P<0.05) late in the fast (0.81±0.06 vs 0.50±0.03 mg/dl per min). This resulted in mean blood glucose returning to baseline by 120 min in early fasted seals versus more than 150 min in late-fasted seals (Fig. 1) consistent with impaired clearance of infused glucose in the latter. The mean AUCglucose was almost doubled in response to the GTT and glucose remained significantly elevated in late-fasted animals (Fig. 1 insert), the findings that coincide with the slower clearance of glucose.

Figure 1
Figure 1

Mean (±s.e.m.) percent change from baseline of blood glucose during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. +, *, #, and ‡ denote significant difference from baseline (time 0) at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. a, b, and c denote significant difference from early fasting at P<0.05, P<0.01, and P<0.001 respectively.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Hormones

Mean percent change in insulin significantly increased at 15 min post-infusion in both groups; however, the increase in mean insulin at 30 min was 2.5-fold greater in early fasted seals (Fig. 2A). In contrast to early fasted animals, which returned to baseline after 120 min, the mean insulin remained elevated throughout the sampling period in late-fasted animals, coinciding with the elevated blood glucose at the same time point in the late group, suggesting that the impaired clearance of glucose maintained chronic stimulation of insulin (Fig. 2A). Mean AUCinsulin did not differ between the two groups (Fig. 2A insert). GTTs induced similar suppressive effects on plasma cortisol during the early and late periods, with the mean percent change in plasma cortisol decreasing (P<0.05) until 120 min post-infusion in both early- and late-fasted animals (Fig. 2B). Mean AUCcortisol was not different between the two groups suggesting that the adrenal responsiveness to a glucose challenge was not altered with fasting duration (Fig. 2B insert). Both early- and late-fasted seals also showed similar responses to GTT with respect to plasma adiponectin, which decreased (P<0.05) and did not fully recover until after 150 min (Fig. 3). Plasma GLP-1, IGF1, and leptin did not change in response to the GTT.

Figure 2
Figure 2

Mean (±s.e.m.) percent change from baseline of (A) plasma insulin and (B) plasma cortisol during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. +, *, #, and ‡ denote significant difference from baseline (time 0) at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. a, and d denote significant difference from early fasting at P<0.05 and P<0.0001 respectively.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Figure 3
Figure 3

Mean (±s.e.m.) percent change from baseline of plasma adiponectin during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. + and ‡ denote significant difference from baseline (time 0) at P<0.05 and P<0.0001 respectively.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Non-esterified fatty acids

Plasma NEFAs decreased (P<0.0001) in response to the GTT early in the fast and remain suppressed until 150 min post-infusion (Fig. 4). In contrast, plasma NEFAs increased (P<0.0001) in response to GTTs in late-fasted animals and levels remained elevated until 120 min post-infusion (Fig. 4).

Figure 4
Figure 4

Mean (±s.e.m.) percent change from baseline of plasma NEFA during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. + and ‡ denote significant difference from baseline (time 0) at P<0.5 and P<0.0001 respectively. d denotes significant difference from early fasting at P<0.0001.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Intracellular proteins

No significant changes were observed in the expression of total IR, IRS-1, AKT2, AMPK, Glut4, or VAMP2 in response to the GTT during either period. Phosphorylated IR increased about twofold (P<0.0001) at 60 min but returned to basal levels by 150 min in the early fasted animals (Fig. 5A). In the late-fasted seals, phosphorylated IR increased 22% (P<0.01) and not until 150 min in response to glucose infusion (Fig. 5A). Tyrosine phosphorylated IRS-1 increased 80% (P<0.0001) by 60 min and returned to basal levels by 150 min early in the fast (Fig. 5B). Tyrosine phosphorylated IRS-1 increased 20% (P<0.05) at 150 min (Fig. 5B). PI3k expression did not change in the early fasted animals but increased 20% (P<0.05) at 150 min in the late-fasted animals (Fig. 5C). Phosphorylated AKT2 increased 50% (P<0.001) in response to the GTT in early fasted animals; however, the recovery at 150 min overcompensated and levels were reduced 24% (P<0.01) compared with basal levels (Fig. 5C). Phosphorylated AKT2 increased 40% (P<0.001) in late-fasted seals at 150 min (Fig. 4C) and levels were greater (P<0.0001) than early at the same time point (Fig. 5D). Phosphorylated AMPK did not change in response to GTT early in the fast but increased 60% (P<0.001) at 60 min and remained elevated (P<0.0001) at 150 min in the late-fasted animals (Fig. 6A). Similar to AMPK, PPARγ expression did not change as a result of GTT in the early fasted seals, but PPARγ expression increased 75% (P<0.0001) at 60 min and remained elevated (P<0.001) at 150 min in the late-fasted seals (Fig. 6B).

Figure 5
Figure 5

Mean (±s.e.m.) ratios and representative western blots of (A) p-IR to IR, (B) p-IRS-1 to IRS-1, (C) PI3k to β-actin, and (D) p-AKT2 to AKT2 in adipose tissue of early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups at 0, 60, and 150 min post-infusion. +, *, #, and ‡ denote significant difference from time 0 at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. d denotes significant difference from early fasting at P<0.0001.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Figure 6
Figure 6

Mean (±s.e.m.) ratios and representative western blots of (A) p-AMPK to AMPK and (B) PPARγ to lamin in adipose tissue of early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups at 0, 60, and 150 min post-infusion. # and ‡ denote significant difference from time 0 at P<0.001 and P<0.0001 respectively. c and d denote significant difference from early fasting at P<0.001 and P<0.0001 respectively.

Citation: Journal of Endocrinology 209, 3; 10.1530/JOE-11-0017

Discussion

Insulin plays a critical role in regulating the availability of metabolic substrates, and defects in insulin signaling in one tissue can have profound effects on whole-body insulin sensitivity. In this study, we found significant reductions in plasma insulin and adipose tissue insulin signaling, leading to an impaired response to a GTT in late-fasted elephant seal pups. The impaired response, associated with an increase in adipose tissue AMPK activity, suggests that AMPK 1) is activated following the onset of insulin resistance and 2) may be compensating for the reduction in cellular insulin signaling. Because the contributions of AMPK activation to the regulation of glucose during prolonged fasting and insulin resistance are not well defined, this study enhances our understanding of the cellular signaling events and mechanisms used by prolong-fasted mammals to counteract the deleterious effects of fasting-induced insulin resistance. In addition, this study provides a unique opportunity to assess both the cellular and the systemic responses to a GTT in a mammal adapted to insulin resistance.

Although the GTT stimulated activation of the insulin signaling pathway at both periods, the critical difference is the timing of the events. In early fasted animals, GTT stimulated phosphorylation of insulin receptor, IRS-1, and AKT2 at 60 min post-infusion, but phosphorylation of these insulin signaling proteins is not increased until 150 min in late-fasted seals, suggesting that delayed phosphorylation of these proteins contributes to the insulin-resistant condition observed. In addition to the delayed phosphorylation of these signaling proteins, the magnitude of the phosphorylation in late-fasted animals is much less than in early fasted animals, suggesting that, in addition to the timing of phosphorylation, blunted phosphorylation also contributes to the observed insulin resistance.

The similarities in AUCinsulin following the GTT would suggest that glucose-stimulated pancreatic secretion of insulin is not impaired over the measurement period (150 min); however, the difference in the secretion pattern over time is indicative of differential pancreatic responsiveness that is likely a residual and characteristic effect of fasting duration. In early fasted animals, the patterns of insulin secretion and signaling and glucose clearance are more characteristic of healthy, non-insulin-resistant mammals. Unlike early fasted animals in which plasma insulin returned to baseline levels (at 120 min), plasma insulin plateaued after the peak secretion at 30 min, and stayed elevated over the measurement period, coinciding with reduced glucose clearance suggesting that the chronically elevated blood glucose levels in late-fasted animals maintained chronic, however blunted, stimulation of the pancreas. This pancreatic response is a unique observation in that insulin-resistant mammals (e.g. humans, rodents, and canines) usually maintain chronically elevated plasma insulin at much higher concentrations (McGuinness et al. 1990, Fernandez-Veledo et al. 2008, Shanik et al. 2008) and also suggests that pancreatic sensitivity to glucose is impaired in late-fasted animals.

Glucocorticoids inhibit pancreatic insulin secretion, inhibit glucose uptake in peripheral tissues, and stimulate hepatic gluconeogenesis and free fatty acid release from adipocytes (Billaudel & Sutter 1979, Khan et al. 1992, Lambilotte et al. 1997, Khani & Tayek 2001, Macfarlane et al. 2008, van Raalte 2009). This is generally assumed to represent an adaptive response in animals during stressful conditions (e.g. nutritional stress and sustained exercise) to ensure that a pool of metabolic substrates is maintained in circulation and available to supply increased energetic demands. Although plasma cortisol increased nearly twofold late in the fast, GTTs suppressed plasma cortisol similarly between both the periods suggesting that 1) the adrenal gland (zona fasciculata) is similarly responsive to negative feedback regulation of glucocorticoid secretion and 2) fasting duration has not altered the adrenal's sensitivity to glucose (substrate availability). Furthermore, the integrated (AUCinsulin) GTT-induced increase of insulin was similar between both the periods and coincided with similar degrees of suppression in cortisol during the GTTs, suggesting that cortisol contributes to the suppression of insulin secretion during prolonged fasting.

In addition to elevated cortisol, free fatty acids can also impair insulin signaling in peripheral tissues and may be the link by which dyslipidemia contributes to insulin resistance (Golay et al. 1987, Epps-Fung et al. 1997, Kashyap et al. 2003). Furthermore, data regarding the response of NEFAs to GTTs is scarce (Sumner et al. 2004) and not well described under prolonged fasting conditions in models of insulin resistance. A unique observation of this study was the differential response of NEFAs to a glucose infusion represented by suppression of NEFAs early and stimulation late. This differential NEFA response to GTTs is likely an evolutionary adaptation associated with resource allocation of substrates necessary to maintain energetic demands associated with prolonged fasting (conservation of lipid stores early in the fast, mobilization of lipid stores late in the fast when metabolism has shifted to oxidation of fat). Adiponectin promotes insulin sensitivity in peripheral tissues by stimulating the removal of free fatty acids (FFA) from circulation and thus its decrease is another commonly used measure to gauge insulin resistance (Punyadeera et al. 2005, Ahima 2006, Ahima & Lazar 2008). Though the precise mechanisms have not been elucidated, we propose that suppressed adiponectin resulting from the GTTs, along with the increased availability of glucose, suppressed fatty acid oxidation and increased lipolysis, thus increasing plasma NEFAs. Moreover, the increase in the expression of PPARγ following the infusion in late-fasted animals reinforces the suggestion that lipolysis was increased (Benton et al. 2010, Hsiao et al. 2011), contributing to the increased FFA release.

Though Glut4 is considered to be an insulin-responsive glucose transporter, AMPK has been shown to stimulate Glut4 translocation in a number of tissues, including adipose tissue (Hardie 2003, Yamaguchi et al. 2005). Despite the reduced phosphorylation of insulin receptor, IRS-1, and AKT late in the fast, the phosphorylation of AMPK increased and may have compensated for the suppressed insulin signaling. The observed increase in the expression of Glut4 and VAMP2 late in the fast may be adaptive to compensate for the elevated NEFAs associated with late fasting and necessary to maintain proper regulation of glucose uptake in adipose tissue. Increased Glut4 expression may have resulted from increased fatty acid oxidation late in the seal's fast as ligands (such as palmitoyl l-carnitine) derived from mitochondrial fatty acid import have been shown to increase Glut4 mRNA expression (Griesel et al. 2010). If the increased availability of glucose inhibited fatty acid oxidation, it could have also triggered the activation of AMPK to compensate for the suppression of insulin signaling by FFA and increase Glut4 turnover, thus helping to restore proper energy balance in adipocytes.

In conclusion, the sustained elevation of blood glucose (compared to its baseline concentration) following the infusion late in the seal's fast demonstrates that glucose clearance is impaired and is indicative of insulin resistance. The nearly twofold increase in plasma cortisol with fasting supports the idea that glucocorticoids suppress insulin secretion late in the seal's fast and potentially contributed to the decreased clearance of blood glucose. The suppression of adiponectin paired with a prominent increase in NEFA following the infusion in late fasted animals further reinforces the idea that prolonged fasting induces insulin resistance in the seal. Because FFAs inhibit insulin-mediated glucose uptake, and therefore decrease insulin sensitivity, increased plasma NEFAs in response to GTTs provide a probable cause for the diminished insulin signaling in late-fasted seals. Determining how the seal is able to maintain such high levels of FFA and cortisol, without any apparent detriment to its health, would be of great benefit to our understanding of the conditions that manifest insulin resistance and type 2 diabetes mellitus (T2DM). In addition, increased AMPK activity following the glucose infusion supports the suggestion that AMPK stimulated Glut4 translocation, in the absence of normal insulin signaling, to restore energy balance in adipocytes. If AMPK functions in the manner that we suggest, determining how it can be readily activated in the elephant seal will improve our understanding of the mechanisms possessed by mammals adapted to chronic food deprivation to regulate metabolism during prolonged fasting and may also lead to improvements to the current treatments for insulin resistance and T2DM.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by a grant from the National Institutes of Health (NIH NHLBI R01HL91767 (R M O, D E C)) and a Research Supplement to NIH NHLBI R01HL91767 (R M O).

Acknowledgements

We would like to thank Dr D P Costa (UCSC) for providing laboratory space and J Minas, R Rodriguez, and J P Vázquez-Medina for their assistance in the field and with laboratory analyses. We thank Dr B A Horwitz for her comments on a draft of the manuscript. We also thank Año Nuevo State Park for logistic support of this work.

References

  • AdamsSHCostaDP1993Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast. Journal of Comparative Physiology B: Biochemical Systemic and Environmental Physiology163367373doi:10.1007/BF00265640.

    • Search Google Scholar
    • Export Citation
  • AhimaRS2006Adipose tissue as an endocrine organ. Obesity14242S249Sdoi:10.1038/oby.2006.317.

  • AhimaRSLazarMA2008Adipokines and the peripheral and neural control of energy balance. Molecular Endocrinology2210231031doi:10.1210/me.2007-0529.

    • Search Google Scholar
    • Export Citation
  • ArafatAMöhligMWeickertMSchöflCSprangerJPfeifferA2010Improved insulin sensitivity, preserved beta cell function and improved whole-body glucose metabolism after low-dose growth hormone replacement therapy in adults with severe growth hormone deficiency: a pilot study. Diabetologia5313041313doi:10.1007/s00125-010-1738-4.

    • Search Google Scholar
    • Export Citation
  • BentonCRHollowayGPHanX-XYoshidaYSnookLALallyJGlatzJFCLuikenJJFPChabowskiABonenA2010Increased levels of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) improve lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean and insulin-resistant obese Zucker rats. Diabetologia5320082019doi:10.1007/s00125-010-1773-1.

    • Search Google Scholar
    • Export Citation
  • BillaudelBSutterBC1979Direct effect of corticosterone upon insulin secretion studied by three different techniques. Hormone and Metabolic Research11555560doi:10.1055/s-0028-1092779.

    • Search Google Scholar
    • Export Citation
  • CampbellRK2009Type 2 diabetes: where we are today: an overview of disease burden, current treatments, and treatment strategies. Journal of the American Pharmacists Association49S3S9doi:10.1331/JAPhA.2009.09077.

    • Search Google Scholar
    • Export Citation
  • CastelliniMACostaDP1990Relationships between plasma ketones and fasting duration in neonatal elephant seals. American Journal of Physiology. Regulatory Integrative and Comparative Physiology259R1086R1089.

    • Search Google Scholar
    • Export Citation
  • CastelliniMACostaDPHuntleyAC1987Fatty acid metabolism in fasting elephant seal pups. Journal of Comparative Physiology. B Biochemical Systemic and Environmental Physiology157445449doi:10.1007/BF00691828.

    • Search Google Scholar
    • Export Citation
  • CefaluW2010The physiologic role of incretin hormones: clinical applications. Journal of the American Osteopathic Association110S8S14.

    • Search Google Scholar
    • Export Citation
  • ChampagneCDHouserDSCrockerDE2005Glucose production and substrate cycle activity in a fasting adapted animal, the northern elephant seal. Journal of Experimental Biology208859868doi:10.1242/jeb.01476.

    • Search Google Scholar
    • Export Citation
  • CornierMABessesenDHGurevichILeitnerJWDrazninB2006Nutritional upregulation of p85α expression is an early molecular manifestation of insulin resistance. Diabetologia49748754doi:10.1007/s00125-006-0148-0.

    • Search Google Scholar
    • Export Citation
  • CostaDPOrtizCL1982Blood chemistry homeostasis during prolonged fasting in the northern elephant seal. American Journal of Physiology242R591R595.

    • Search Google Scholar
    • Export Citation
  • CzechMP1995Molecular actions of insulin on glucose transport. Annual Review of Nutrition15441471doi:10.1146/annurev.nu.15.070195.002301.

    • Search Google Scholar
    • Export Citation
  • Epps-FungMVWillifordJWellsAHardyRW1997Fatty acid-induced insulin resistance in adipocytes. Endocrinology13843384345doi:10.1210/en.138.10.4338.

    • Search Google Scholar
    • Export Citation
  • Fernandez-VeledoSNieto-VazquezIde CastroJRamosMPBruderleinSMollerPLorenzoM2008Hyperinsulinemia induces insulin resistance on glucose and lipid metabolism in a human adipocytic cell line: paracrine interaction with myocytes. Journal of Clinical Endocrinology and Metabolism9328662876doi:10.1210/jc.2007-2472.

    • Search Google Scholar
    • Export Citation
  • FowlerMAChampagneCDHouserDSCrockerDE2008Hormonal regulation of glucose clearance in lactating northern elephant seals (Mirounga angustirostris). Journal of Experimental Biology21129432949doi:10.1242/jeb.018176.

    • Search Google Scholar
    • Export Citation
  • GolayASwislockiALMChenYReavenGM1987Relationships between plasma-free fatty acid concentration, endogenous glucose production, and fasting hyperglycemia in normal and non-insulin-dependent diabetic individuals. Metabolism36692696doi:10.1016/0026-0495(87)90156-9.

    • Search Google Scholar
    • Export Citation
  • GregoireFMSmasCMSulHS1998Understanding adipocyte differentiation. Physiological Reviews78783809.

  • GrieselBAWeemsJRussellRAAbelEDHumphriesKOlsonAL2010Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling. Diabetes59800807doi:10.2337/db09-1542.

    • Search Google Scholar
    • Export Citation
  • HardieDG2003Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology14451795183doi:10.1210/en.2003-0982.

    • Search Google Scholar
    • Export Citation
  • HsiaoGChapmanJOfrecioJWilkesJResnikJThaparDSubramaniamSSearsD2011Multi-tissue, selective PPAR{gamma} modulation of insulin sensitivity and metabolic pathways in obese rats. American Journal of Physiology. Endocrinology and Metabolism300E164E174doi:10.1152/ajpendo.00219.2010.

    • Search Google Scholar
    • Export Citation
  • KahnBBFlierJS2000Obesity and insulin resistance. Journal of Clinical Investigation106473481doi:10.1172/JCI10842.

  • KashyapSBelfortRGastaldelliAPratipanawatrTBerriaRPratipanawatrWBajajMMandarinoLDeFronzoRCusiK2003A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes5224612474doi:10.2337/diabetes.52.10.2461.

    • Search Google Scholar
    • Export Citation
  • KeithEOOrtizCL1988Glucose kinetics in neonatal elephant seals during postweaning aphagia. Marine Mammal Science599115doi:10.1111/j.1748-7692.1989.tb00326.x.

    • Search Google Scholar
    • Export Citation
  • KhanAOstensonCGBerggrenPOEfendicS1992Glucocorticoid increases glucose cycling and inhibits insulin release in pancreatic islets of ob/ob mice. American Journal of Physiology263E663E666.

    • Search Google Scholar
    • Export Citation
  • KhaniSTayekJA2001Cortisol increases gluconeogenesis in humans: its role in the metabolic syndrome. Clinical Science101739747doi:10.1042/CS20010180.

    • Search Google Scholar
    • Export Citation
  • KirbyVLOrtizCL1994Hormones and fuel regulation in fasting elephant seals. In Elephant Seals: Population Ecology Behavior and Physiolgy pp 374386. Eds Le BoeufBJLawsRM. Berkeley and Los Angeles: University of California Press.

    • Search Google Scholar
    • Export Citation
  • KnopFKVilsbøllTHøjbergPVLarsenSMadsbadSVølundAHolstJJKrarupT2007Reduced incretin effect in type 2 diabetes: cause or consequence of the diabetic state?Diabetes5619511959doi:10.2337/db07-0100.

    • Search Google Scholar
    • Export Citation
  • KouidhiSJarbouiSMarrakchiRClerget FroidevauxMSSeugnetIAbidHBchirFBrahemMDemeneixBGuissoumaH2010Adiponectin expression and metabolic markers in obesity and type 2 diabetes. Journal of Endocrinological InvestigationIn press.

    • Search Google Scholar
    • Export Citation
  • KrassasG2003Endocrine abnormalities in anorexia nervosa. Pediatric Endocrinology Reviews14654.

  • LamTKTWerveGVDGiaccaA2003Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites. American Journal of Physiology. Endocrinology and Metabolism284E281E290doi:10.1152/ajpendo.00332.2002.

    • Search Google Scholar
    • Export Citation
  • LambilotteCGilonPHenquinJC1997Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. Journal of Clinical Investigation99414423doi:10.1172/JCI119175.

    • Search Google Scholar
    • Export Citation
  • LeneySETavareJM2009The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets. Journal of Endocrinology203118doi:10.1677/JOE-09-0037.

    • Search Google Scholar
    • Export Citation
  • LinYSunZ2010Current views on type 2 diabetes. Journal of Endocrinology204111doi:10.1677/JOE-09-0260.

  • MacfarlaneDPForbesSWalkerBR2008Glucocorticoids and fatty acid metabolism in humans: fuelling fat redistribution in the metabolic syndrome. Journal of Endocrinology197189204doi:10.1677/JOE-08-0054.

    • Search Google Scholar
    • Export Citation
  • McGuinnessOPFriedmanACherringtonAD1990Intraportal hyperinsulinemia decreases insulin-stimulated glucose uptake in the dog. Metabolism39127132doi:10.1016/0026-0495(90)90064-J.

    • Search Google Scholar
    • Export Citation
  • MotheIvan ObberghenE1996Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. Journal of Biological Chemistry2711122211227doi:10.1074/jbc.271.19.11222.

    • Search Google Scholar
    • Export Citation
  • OrtizRMWadeCEOrtizCL2001Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups. American Journal of Physiology. Regulatory Integrative and Comparative Physiology280R790R795.

    • Search Google Scholar
    • Export Citation
  • OrtizRMNorenDPOrtizCLTalamantesF2003GH and ghrelin increase with fasting in a naturally adapted species, the northern elephant seal (Mirounga angustirostris). Journal of Endocrinology178533539doi:10.1677/joe.0.1780533.

    • Search Google Scholar
    • Export Citation
  • PerseghinGPetersenKShulmanGI2003Cellular mechanism of insulin resistance: potential links with inflammation. International Journal of Obesity27S6S11doi:10.1038/sj.ijo.0802491.

    • Search Google Scholar
    • Export Citation
  • PunyadeeraCZorencAHGKoopmanRMcAinchAJSmitEMandersRKeizerHACameron-SmithDLoonLJCv2005The effects of exercise and adipose tissue lipolysis on plasma adiponectin concentration and adiponectin receptor expression in human skeletal muscle. European Journal of Endocrinology152427436doi:10.1530/eje.1.01872.

    • Search Google Scholar
    • Export Citation
  • van RaalteDH2009Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options?European Journal of Clinical Investigation398193doi:10.1111/j.1365-2362.2008.02067.x.

    • Search Google Scholar
    • Export Citation
  • RussellRRBergeronRShulmanGIYoungLH1999Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. American Journal of Physiology. Heart and Circulatory Physiology277H643H649.

    • Search Google Scholar
    • Export Citation
  • SaboureauMBobetJBoissinJ1980Cyclic activity of adrenal function and seasonal variations of cortisol peripheral metabolism in a hibernating mammal, the hedgehog (Erinaceus europaeus L.). Journal de Physiologie76617629.

    • Search Google Scholar
    • Export Citation
  • ScheenACastilloMLefèbvreP1988Insulin sensitivity in anorexia nervosa: a mirror image of obesity?Diabetes/Metabolism Reviews4681690doi:10.1002/dmr.5610040705.

    • Search Google Scholar
    • Export Citation
  • ShanikMHXuYŠkrhaJDanknerRZickYRothJ2008Insulin resistance and hyperinsulinemia. Diabetes Care31S262S268doi:10.2337/dc08-s264.

  • SumnerAEBergmanRNVegaGLGenoveseDJCochranCSPacakKWatanabeRMBostonRC2004The multiphasic profile of free fatty acids during the intravenous glucose tolerance test is unresponsive to exogenous insulin. Metabolism5312021207doi:10.1016/j.metabol.2004.03.020.

    • Search Google Scholar
    • Export Citation
  • ViscarraJAVazquez-MedinaJPCrockerDEOrtizRM2011Glut4 is upregulated despite decreased insulin signaling during prolonged fasting in northern elephant seal pups. American Journal of Physiology. Regulatory Integrative and Comparative Physiology300R150R154doi:10.1152/ajpregu.00478.2010.

    • Search Google Scholar
    • Export Citation
  • YamaguchiSKatahiraHOzawaSNakamichiYTanakaTShimoyamaTTakahashiKYoshimotoKImaizumiMONagamatsuS2005Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes. American Journal of Physiology. Endocrinology and Metabolism289E643E649doi:10.1152/ajpendo.00456.2004.

    • Search Google Scholar
    • Export Citation

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    Mean (±s.e.m.) percent change from baseline of blood glucose during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. +, *, #, and ‡ denote significant difference from baseline (time 0) at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. a, b, and c denote significant difference from early fasting at P<0.05, P<0.01, and P<0.001 respectively.

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    Mean (±s.e.m.) percent change from baseline of (A) plasma insulin and (B) plasma cortisol during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. +, *, #, and ‡ denote significant difference from baseline (time 0) at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. a, and d denote significant difference from early fasting at P<0.05 and P<0.0001 respectively.

  • View in gallery

    Mean (±s.e.m.) percent change from baseline of plasma adiponectin during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. + and ‡ denote significant difference from baseline (time 0) at P<0.05 and P<0.0001 respectively.

  • View in gallery

    Mean (±s.e.m.) percent change from baseline of plasma NEFA during the GTT in early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups. + and ‡ denote significant difference from baseline (time 0) at P<0.5 and P<0.0001 respectively. d denotes significant difference from early fasting at P<0.0001.

  • View in gallery

    Mean (±s.e.m.) ratios and representative western blots of (A) p-IR to IR, (B) p-IRS-1 to IRS-1, (C) PI3k to β-actin, and (D) p-AKT2 to AKT2 in adipose tissue of early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups at 0, 60, and 150 min post-infusion. +, *, #, and ‡ denote significant difference from time 0 at P<0.05, P<0.01, P<0.001, and P<0.0001 respectively. d denotes significant difference from early fasting at P<0.0001.

  • View in gallery

    Mean (±s.e.m.) ratios and representative western blots of (A) p-AMPK to AMPK and (B) PPARγ to lamin in adipose tissue of early (n=5; 2–3 week post-weaning) and late (n=8; 6–8 week post-weaning)-fasted elephant seal pups at 0, 60, and 150 min post-infusion. # and ‡ denote significant difference from time 0 at P<0.001 and P<0.0001 respectively. c and d denote significant difference from early fasting at P<0.001 and P<0.0001 respectively.

  • AdamsSHCostaDP1993Water conservation and protein metabolism in northern elephant seal pups during the postweaning fast. Journal of Comparative Physiology B: Biochemical Systemic and Environmental Physiology163367373doi:10.1007/BF00265640.

    • Search Google Scholar
    • Export Citation
  • AhimaRS2006Adipose tissue as an endocrine organ. Obesity14242S249Sdoi:10.1038/oby.2006.317.

  • AhimaRSLazarMA2008Adipokines and the peripheral and neural control of energy balance. Molecular Endocrinology2210231031doi:10.1210/me.2007-0529.

    • Search Google Scholar
    • Export Citation
  • ArafatAMöhligMWeickertMSchöflCSprangerJPfeifferA2010Improved insulin sensitivity, preserved beta cell function and improved whole-body glucose metabolism after low-dose growth hormone replacement therapy in adults with severe growth hormone deficiency: a pilot study. Diabetologia5313041313doi:10.1007/s00125-010-1738-4.

    • Search Google Scholar
    • Export Citation
  • BentonCRHollowayGPHanX-XYoshidaYSnookLALallyJGlatzJFCLuikenJJFPChabowskiABonenA2010Increased levels of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) improve lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean and insulin-resistant obese Zucker rats. Diabetologia5320082019doi:10.1007/s00125-010-1773-1.

    • Search Google Scholar
    • Export Citation
  • BillaudelBSutterBC1979Direct effect of corticosterone upon insulin secretion studied by three different techniques. Hormone and Metabolic Research11555560doi:10.1055/s-0028-1092779.

    • Search Google Scholar
    • Export Citation
  • CampbellRK2009Type 2 diabetes: where we are today: an overview of disease burden, current treatments, and treatment strategies. Journal of the American Pharmacists Association49S3S9doi:10.1331/JAPhA.2009.09077.

    • Search Google Scholar
    • Export Citation
  • CastelliniMACostaDP1990Relationships between plasma ketones and fasting duration in neonatal elephant seals. American Journal of Physiology. Regulatory Integrative and Comparative Physiology259R1086R1089.

    • Search Google Scholar
    • Export Citation
  • CastelliniMACostaDPHuntleyAC1987Fatty acid metabolism in fasting elephant seal pups. Journal of Comparative Physiology. B Biochemical Systemic and Environmental Physiology157445449doi:10.1007/BF00691828.

    • Search Google Scholar
    • Export Citation
  • CefaluW2010The physiologic role of incretin hormones: clinical applications. Journal of the American Osteopathic Association110S8S14.

    • Search Google Scholar
    • Export Citation
  • ChampagneCDHouserDSCrockerDE2005Glucose production and substrate cycle activity in a fasting adapted animal, the northern elephant seal. Journal of Experimental Biology208859868doi:10.1242/jeb.01476.

    • Search Google Scholar
    • Export Citation
  • CornierMABessesenDHGurevichILeitnerJWDrazninB2006Nutritional upregulation of p85α expression is an early molecular manifestation of insulin resistance. Diabetologia49748754doi:10.1007/s00125-006-0148-0.

    • Search Google Scholar
    • Export Citation
  • CostaDPOrtizCL1982Blood chemistry homeostasis during prolonged fasting in the northern elephant seal. American Journal of Physiology242R591R595.

    • Search Google Scholar
    • Export Citation
  • CzechMP1995Molecular actions of insulin on glucose transport. Annual Review of Nutrition15441471doi:10.1146/annurev.nu.15.070195.002301.

    • Search Google Scholar
    • Export Citation
  • Epps-FungMVWillifordJWellsAHardyRW1997Fatty acid-induced insulin resistance in adipocytes. Endocrinology13843384345doi:10.1210/en.138.10.4338.

    • Search Google Scholar
    • Export Citation
  • Fernandez-VeledoSNieto-VazquezIde CastroJRamosMPBruderleinSMollerPLorenzoM2008Hyperinsulinemia induces insulin resistance on glucose and lipid metabolism in a human adipocytic cell line: paracrine interaction with myocytes. Journal of Clinical Endocrinology and Metabolism9328662876doi:10.1210/jc.2007-2472.

    • Search Google Scholar
    • Export Citation
  • FowlerMAChampagneCDHouserDSCrockerDE2008Hormonal regulation of glucose clearance in lactating northern elephant seals (Mirounga angustirostris). Journal of Experimental Biology21129432949doi:10.1242/jeb.018176.

    • Search Google Scholar
    • Export Citation
  • GolayASwislockiALMChenYReavenGM1987Relationships between plasma-free fatty acid concentration, endogenous glucose production, and fasting hyperglycemia in normal and non-insulin-dependent diabetic individuals. Metabolism36692696doi:10.1016/0026-0495(87)90156-9.

    • Search Google Scholar
    • Export Citation
  • GregoireFMSmasCMSulHS1998Understanding adipocyte differentiation. Physiological Reviews78783809.

  • GrieselBAWeemsJRussellRAAbelEDHumphriesKOlsonAL2010Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling. Diabetes59800807doi:10.2337/db09-1542.

    • Search Google Scholar
    • Export Citation
  • HardieDG2003Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology14451795183doi:10.1210/en.2003-0982.

    • Search Google Scholar
    • Export Citation
  • HsiaoGChapmanJOfrecioJWilkesJResnikJThaparDSubramaniamSSearsD2011Multi-tissue, selective PPAR{gamma} modulation of insulin sensitivity and metabolic pathways in obese rats. American Journal of Physiology. Endocrinology and Metabolism300E164E174doi:10.1152/ajpendo.00219.2010.

    • Search Google Scholar
    • Export Citation
  • KahnBBFlierJS2000Obesity and insulin resistance. Journal of Clinical Investigation106473481doi:10.1172/JCI10842.

  • KashyapSBelfortRGastaldelliAPratipanawatrTBerriaRPratipanawatrWBajajMMandarinoLDeFronzoRCusiK2003A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes5224612474doi:10.2337/diabetes.52.10.2461.

    • Search Google Scholar
    • Export Citation
  • KeithEOOrtizCL1988Glucose kinetics in neonatal elephant seals during postweaning aphagia. Marine Mammal Science599115doi:10.1111/j.1748-7692.1989.tb00326.x.

    • Search Google Scholar
    • Export Citation
  • KhanAOstensonCGBerggrenPOEfendicS1992Glucocorticoid increases glucose cycling and inhibits insulin release in pancreatic islets of ob/ob mice. American Journal of Physiology263E663E666.

    • Search Google Scholar
    • Export Citation
  • KhaniSTayekJA2001Cortisol increases gluconeogenesis in humans: its role in the metabolic syndrome. Clinical Science101739747doi:10.1042/CS20010180.

    • Search Google Scholar
    • Export Citation
  • KirbyVLOrtizCL1994Hormones and fuel regulation in fasting elephant seals. In Elephant Seals: Population Ecology Behavior and Physiolgy pp 374386. Eds Le BoeufBJLawsRM. Berkeley and Los Angeles: University of California Press.

    • Search Google Scholar
    • Export Citation
  • KnopFKVilsbøllTHøjbergPVLarsenSMadsbadSVølundAHolstJJKrarupT2007Reduced incretin effect in type 2 diabetes: cause or consequence of the diabetic state?Diabetes5619511959doi:10.2337/db07-0100.

    • Search Google Scholar
    • Export Citation
  • KouidhiSJarbouiSMarrakchiRClerget FroidevauxMSSeugnetIAbidHBchirFBrahemMDemeneixBGuissoumaH2010Adiponectin expression and metabolic markers in obesity and type 2 diabetes. Journal of Endocrinological InvestigationIn press.

    • Search Google Scholar
    • Export Citation
  • KrassasG2003Endocrine abnormalities in anorexia nervosa. Pediatric Endocrinology Reviews14654.

  • LamTKTWerveGVDGiaccaA2003Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites. American Journal of Physiology. Endocrinology and Metabolism284E281E290doi:10.1152/ajpendo.00332.2002.

    • Search Google Scholar
    • Export Citation
  • LambilotteCGilonPHenquinJC1997Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. Journal of Clinical Investigation99414423doi:10.1172/JCI119175.

    • Search Google Scholar
    • Export Citation
  • LeneySETavareJM2009The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets. Journal of Endocrinology203118doi:10.1677/JOE-09-0037.

    • Search Google Scholar
    • Export Citation
  • LinYSunZ2010Current views on type 2 diabetes. Journal of Endocrinology204111doi:10.1677/JOE-09-0260.

  • MacfarlaneDPForbesSWalkerBR2008Glucocorticoids and fatty acid metabolism in humans: fuelling fat redistribution in the metabolic syndrome. Journal of Endocrinology197189204doi:10.1677/JOE-08-0054.

    • Search Google Scholar
    • Export Citation
  • McGuinnessOPFriedmanACherringtonAD1990Intraportal hyperinsulinemia decreases insulin-stimulated glucose uptake in the dog. Metabolism39127132doi:10.1016/0026-0495(90)90064-J.

    • Search Google Scholar
    • Export Citation
  • MotheIvan ObberghenE1996Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. Journal of Biological Chemistry2711122211227doi:10.1074/jbc.271.19.11222.

    • Search Google Scholar
    • Export Citation
  • OrtizRMWadeCEOrtizCL2001Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups. American Journal of Physiology. Regulatory Integrative and Comparative Physiology280R790R795.

    • Search Google Scholar
    • Export Citation
  • OrtizRMNorenDPOrtizCLTalamantesF2003GH and ghrelin increase with fasting in a naturally adapted species, the northern elephant seal (Mirounga angustirostris). Journal of Endocrinology178533539doi:10.1677/joe.0.1780533.

    • Search Google Scholar
    • Export Citation
  • PerseghinGPetersenKShulmanGI2003Cellular mechanism of insulin resistance: potential links with inflammation. International Journal of Obesity27S6S11doi:10.1038/sj.ijo.0802491.

    • Search Google Scholar
    • Export Citation
  • PunyadeeraCZorencAHGKoopmanRMcAinchAJSmitEMandersRKeizerHACameron-SmithDLoonLJCv2005The effects of exercise and adipose tissue lipolysis on plasma adiponectin concentration and adiponectin receptor expression in human skeletal muscle. European Journal of Endocrinology152427436doi:10.1530/eje.1.01872.

    • Search Google Scholar
    • Export Citation
  • van RaalteDH2009Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options?European Journal of Clinical Investigation398193doi:10.1111/j.1365-2362.2008.02067.x.

    • Search Google Scholar
    • Export Citation
  • RussellRRBergeronRShulmanGIYoungLH1999Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. American Journal of Physiology. Heart and Circulatory Physiology277H643H649.

    • Search Google Scholar
    • Export Citation
  • SaboureauMBobetJBoissinJ1980Cyclic activity of adrenal function and seasonal variations of cortisol peripheral metabolism in a hibernating mammal, the hedgehog (Erinaceus europaeus L.). Journal de Physiologie76617629.

    • Search Google Scholar
    • Export Citation
  • ScheenACastilloMLefèbvreP1988Insulin sensitivity in anorexia nervosa: a mirror image of obesity?Diabetes/Metabolism Reviews4681690doi:10.1002/dmr.5610040705.

    • Search Google Scholar
    • Export Citation
  • ShanikMHXuYŠkrhaJDanknerRZickYRothJ2008Insulin resistance and hyperinsulinemia. Diabetes Care31S262S268doi:10.2337/dc08-s264.

  • SumnerAEBergmanRNVegaGLGenoveseDJCochranCSPacakKWatanabeRMBostonRC2004The multiphasic profile of free fatty acids during the intravenous glucose tolerance test is unresponsive to exogenous insulin. Metabolism5312021207doi:10.1016/j.metabol.2004.03.020.

    • Search Google Scholar
    • Export Citation
  • ViscarraJAVazquez-MedinaJPCrockerDEOrtizRM2011Glut4 is upregulated despite decreased insulin signaling during prolonged fasting in northern elephant seal pups. American Journal of Physiology. Regulatory Integrative and Comparative Physiology300R150R154doi:10.1152/ajpregu.00478.2010.

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