Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.

Role of AMP-activated protein kinase in exercise capacity, whole body glucose homeostasis, and glucose transport in skeletal muscle -insight from analysis of a transgenic mouse model-.

To examine the role of muscle AMP-activated protein kinase (AMPK) in maximal exercise capacity, whole body glucose homeostasis, and glucose transport in skeletal muscle, we generated muscle-specific transgenic mice carrying cDNAs of inactive AMPK alpha2 (alpha2i TG). Fed blood glucose was slightly higher in alpha2i TG mice compared to wild type littermates, however, the difference was not statistically significant. In alpha2i TG mice, glucose tolerance was slightly impaired in male, but not in female mice, compared to wild type littermates. Maximal exercise capacity was dramatically reduced in alpha2i TG mice, suggesting that AMPK alpha2 has a critical role in skeletal muscle during exercise. We confirmed that known insulin-independent stimuli of glucose transport including mitochondrial respiration inhibition, hyperosmolarity, and muscle contraction increased both AMPK alpha1 and alpha2 activities in isolated EDL muscle in wild type mice. While, alpha2 activation was severely blunted and alpha1 activation was only slightly reduced in alpha2i TG mice by these insulin independent stimuli compared to wild type mice. Mitochondrial respiration inhibition-induced glucose transport was fully inhibited in isolated EDL muscles in alpha2i TG mice. However, contraction- or hyperosmolarity-induced glucose transport was nearly normal. These results suggest that AMPK alpha2 activation is essential for some, but not all insulin-independent glucose transport.

High-fiber oat cereal compared with wheat cereal consumption favorably alters LDL-cholesterol subclass and particle numbers in middle-aged and older men.

BACKGROUND: No studies have examined whether increased consumption of oat cereal, rich in soluble fiber, favorably alters lipoprotein particle size and number. OBJECTIVE: We examined the effects of large servings of either oat or wheat cereal on plasma lipids, lipoprotein subclasses, lipoprotein particle diameters, and LDL particle number. DESIGN: Thirty-six overweight men aged 50-75 y were randomly assigned to consume daily for 12 wk either oat or wheat cereal providing 14 g dietary fiber/d. Before and after the intervention, plasma lipid and lipoprotein subclasses were measured with proton nuclear magnetic resonance spectroscopy, and whole-body insulin sensitivity was estimated with the frequently sampled intravenous-glucose-tolerance test. RESULTS: Time-by-treatment interactions (P < 0.05) for LDL cholesterol (oat: -2.5%; wheat: 8.0%), small LDL cholesterol (oat: -17.3%; wheat: 60.4%), LDL particle number (oat: -5.0%; wheat: 14.2%), and LDL:HDL cholesterol (oat: -6.3%; wheat: 14.2%) were observed. Time-by-treatment interactions were nearly significant for total cholesterol (oat: -2.5%; wheat: 6.3%; P = 0.08), triacylglycerol (oat: -6.6%; wheat: 22.0%; P = 0.07), and VLDL triacylglycerol (oat: -7.6%; wheat: 2.7%; P = 0.08). No significant time-by-treatment interactions were observed for HDL cholesterol, HDL-cholesterol subclasses, or LDL, HDL, and VLDL particle diameters. Insulin sensitivity did not change significantly with either intervention. CONCLUSIONS: The oat compared with the wheat cereal produced lower concentrations of small, dense LDL cholesterol and LDL particle number without producing adverse changes in blood triacylglycerol or HDL-cholesterol concentrations. These beneficial alterations may contribute to the cardioprotective effect of oat fiber.

Whole-body insulin sensitivity, low-density lipoprotein (LDL) particle size, and oxidized LDL in overweight, nondiabetic men.

Insulin resistance is often accompanied by elevated plasma triglycerides (TG) and a preponderance of small, dense low-density lipoprotein (LDL) particles. However, it remains unclear whether or not insulin resistance is related to LDL particle size, independent of plasma TG. We sought to determine the strength of the relationships among these variables in a group of overweight, nondiabetic men (N = 34; body mass index [BMI], 25 to 35 kg/m(2); age, 50 to 75 years), as well as to examine the possible relation between insulin sensitivity and oxidized LDL (oxLDL). We also examined the strength of the relationships between these lipid variables and estimates of insulin sensitivity using calculated indices based on fasting insulin and glucose concentrations. Insulin sensitivity (Si) was significantly associated with total TG (r = -0.61, P <.001), very-low-density lipoprotein (VLDL)-TG (r = -0.60, P <.001), and LDL size (r =.414, P <.05). LDL size was also significantly associated with TG (r = -0.73, P <.001), VLDL-TG (r = -0.73, P <.001), high-density lipoprotein-cholesterol (HDL-C) (r = 0.65, P <.001), the quantitative insulin sensitivity check index (QUICKI) (rho = 0.46, P <.01), and the homeostatic model for the assessment of insulin resistance (HOMA-IR) (rho = -0.45, P <.01). Si was a significant predictor of LDL size, with age and BMI also independent contributors to the variance in LDL size (R(2) = 0.172). However, when TG and HDL-C were added to the model, Si was no longer a significant predictor of LDL size. The correlation between Si and oxLDL was weak, but stastically significant (rho = -0.40, P =.02). These data suggest that the relation between Si and LDL size is largely mediated by plasma TG, and that Si is only weakly related to oxLDL in overweight, nondiabetic men.

Ablation of AMP-activated protein kinase alpha2 activity exacerbates insulin resistance induced by high-fat feeding of mice.

OBJECTIVE: We determined whether muscle AMP-activated protein kinase (AMPK) has a role in the development of insulin resistance. RESEARCH DESIGN AND METHODS: Muscle-specific transgenic mice expressing an inactive form of the AMPK alpha2 catalytic subunit (alpha2i TG) and their wild-type littermates were fed either a high-fat (60% kcal fat) or a control (10% kcal fat) diet for 30 weeks. RESULTS: Compared with wild-type mice, glucose tolerance in alpha2i TG mice was slightly impaired on the control diet and significantly impaired on the high-fat diet. To determine whether the whole-body glucose intolerance was associated with impaired insulin sensitivity in skeletal muscle, glucose transport in response to submaximal insulin (450 microU/ml) was measured in isolated soleus muscles. On the control diet, insulin-stimulated glucose transport was reduced by approximately 50% in alpha2i TG mice compared with wild-type mice. High-fat feeding partially decreased insulin-stimulated glucose transport in wild-type mice, while high-fat feeding resulted in a full blunting of insulin-stimulated glucose transport in the alpha2i TG mice. High-fat feeding in alpha2i TG mice was accompanied by decreased expression of insulin signaling proteins in gastrocnemius muscle. CONCLUSIONS: The lack of skeletal muscle AMPK alpha2 activity exacerbates the development of glucose intolerance and insulin resistance caused by high-fat feeding and supports the thesis that AMPK alpha2 is an important target for the prevention/amelioration of skeletal muscle insulin resistance through lifestyle (exercise) and pharmacologic (e.g., metformin) treatments.

Dissociation of AMP-activated protein kinase and p38 mitogen-activated protein kinase signaling in skeletal muscle.

AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle. The p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. Here we used several different models of altered AMPK activity to determine whether p38 MAPK is a downstream intermediate of AMPK-mediated signaling in skeletal muscle. First, L6 myoblasts and myotubes were treated with AICAR, an AMPK stimulator. AMPK phosphorylation was significantly increased, but there was no change in p38 MAPK phosphorylation. Similarly, AICAR incubation of isolated rat extensor digitorum longus (EDL) muscles did not increase p38 phosphorylation. Next, we used transgenic mice expressing an inactive form of the AMPKalpha2 catalytic subunit in skeletal muscle (AMPKalpha2i TG mice). AMPKalpha2i TG mice did not exhibit any defect in basal or contraction-induced p38 MAPK phosphorylation. We also used transgenic mice expressing an activating mutation in the AMPKgamma1 subunit (gamma1R70Q TG mice). Despite activated AMPK, basal p38 MAPK phosphorylation was not different between wild type and gamma1R70Q TG mice. In addition, muscle contraction-induced p38 MAPK phosphorylation was significantly blunted in the gamma1R70Q TG mice. In conclusion, increasing AMPK activity by AICAR and AMPKgamma1 mutation does not increase p38 MAPK phosphorylation in skeletal muscle. Furthermore, AMPKalpha2i TG mice lacking contraction-stimulated AMPK activity have normal p38 MAPK phosphorylation. These results suggest that p38 MAPK is not a downstream component of AMPK-mediated signaling in skeletal muscle.

Regulation of dishevelled and beta-catenin in rat skeletal muscle: an alternative exercise-induced GSK-3beta signaling pathway.

beta-catenin is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. beta-Catenin is activated upon its dephosphorylation, an event triggered by Dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). In skeletal muscle, both insulin and exercise decrease GSK-3beta activity, and we tested the hypothesis that these two stimuli regulate beta-catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3beta, and beta-catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased beta-catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. beta-Catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (r(2) = 0.84, P < 0.001). beta-Catenin dephosphorylation was accompanied by increases in GSK-3beta Ser(9) phosphorylation and Dvl-GSK-3beta association. In contrast to exercise, maximal insulin treatment (1 U/kg body wt) had no effect on skeletal muscle beta-catenin phosphorylation or Dvl-GSK-3beta interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3beta in skeletal muscle, an event paralleled by beta-catenin dephosphorylation.

Circulating tumor necrosis factor alpha is higher in non-obese, non-diabetic Mexican Americans compared to non-Hispanic white adults.

Mexican Americans (MA) exhibit high risk for the insulin resistance syndrome characterized by subclinical inflammation and greater risk for type 2 diabetes compared with non-Hispanic white (NHW) adults. The reasons for this phenomenon remain obscure. Because the inflammatory cytokine, tumor necrosis factor-alpha (TNF alpha), is associated with insulin resistance in various models of obesity and diabetes, we sought to determine whether circulating concentrations of this cytokine and its soluble receptors are higher in MA than NHW, and also to determine if the TNF alpha system is related to the lower insulin sensitivity in MA. Fasting blood samples were used to determine concentrations of TNF alpha, soluble TNF receptors 1 (sTNFR1) and 2 (sTNFR2) in the same 13 MA (7 women, 6 men, age=27.0+/-2.0 years, BMI=23.0+/-0.7) and 13 NHW (7 women, 6 men, age=24.8+/-1.5 years, BMI=22.8+/-0.6) previously shown to exhibit differences in insulin sensitivity. Circulating TNF alpha was significantly higher (3.11+/-0.38 vs. 2.10+/-0.24 pg/ml, p<0.05) and sTNFR2 was significantly lower (1324+/-85 vs. 1925+/-127 pg/ml, p<0.05) among MA compared with NHW subjects. Soluble TNFR1 was not different between groups (MA: 970+/-111 pg/ml vs. NHW: 1218+/-73 pg/ml, p=0.07). TNF alpha, sTNFR1 and sTNFR2 were not correlated with HOMA-IR when the two groups were analyzed in aggregate. This study documents higher circulating TNF alpha concentrations in non-obese, non-diabetic MA, a population group at increased risk for the metabolic syndrome and the untoward effects of sub-clinical inflammation. The clinical implications of this difference, if any, are not yet known.

AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle.

To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive alpha2 (alpha2i TG) and alpha1 (alpha1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In alpha2i TG mice basal alpha2 activity was barely detectable, whereas basal alpha1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK alpha2 activity in alpha2i TG mice, whereas alpha1 activation was attenuated by only 30-50%. Glucose transport was measured in vitro using isolated EDL muscles from alpha2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in alpha2i TG mice; however, the lack of AMPK alpha2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[(3)H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in alpha2i TG mice. Thus, AMPK alpha2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK alpha2 is one of multiple signaling pathways.

Regulation of IkappaB kinase and NF-kappaB in contracting adult rat skeletal muscle.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor with important roles in regulating innate immune and inflammatory responses. NF-kappaB is activated through the phosphorylation of its inhibitor, IkappaB, by the IkappaB kinase (IKK) complex. Physical exercise elicits changes in skeletal muscle gene expression, yet signaling cascades and transcription factors involved remain largely unknown. To determine whether NF-kappaB signaling is regulated by exercise in vivo, rats were run on a motorized treadmill for 5-60 min. Exercise resulted in up to twofold increases in IKKalpha/beta phosphorylation in the soleus and red gastrocnemius muscles throughout the time course studied. In red gastrocnemius muscles, NF-kappaB activity increased 50% 1-3 h after 60 min of treadmill exercise, returning to baseline by 5 h. Contraction of isolated extensor digitorum longus muscles in vitro increased IKKalpha/beta phosphorylation sevenfold and this was accompanied by a parallel increase in IkappaBalpha phosphorylation. Additional kinases that are activated by exercise include p38, extracellular-signal regulated protein kinase (ERK), and AMP-activated protein kinase (AMPK). Inhibitors of p38 (SB-203580) and ERK (U-0126) blunted contraction-mediated IKK phosphorylation by 39 +/- 4% (P = 0.06) and 35 +/- 10% (P = 0.09), respectively, and in combination by 76 +/- 5% (P < 0.05), suggesting that these kinases might influence the activation of IKK and NF-kappaB during exercise. In contrast, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, an activator of AMPK, had no effect on either IKK or NF-kappaB activity. In conclusion, acute submaximal exercise transiently stimulates NF-kappaB signaling in skeletal muscle. This activation is a local event because it can occur in the absence of exercise-derived systemic factors.

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle.

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.

p38gamma MAPK regulation of glucose transporter expression and glucose uptake in L6 myotubes and mouse skeletal muscle.

Skeletal muscle expresses at least three p38 MAPKs (alpha, beta, gamma). However, no studies have examined the potential regulation of glucose uptake by p38gamma, the isoform predominantly expressed in skeletal muscle and highly regulated by exercise. L6 myotubes were transfected with empty vector (pCAGGS), activating MKK6 (MKK6CA), or p38gamma-specific siRNA. MKK6CA-transfected cells had higher rates of basal 2-deoxy-d-[3H]glucose (2-DG) uptake (P < 0.05) but lower rates of 2,4-dinitrophenol (DNP)-stimulated glucose uptake, an uncoupler of oxidative phosphorylation that operates through an insulin-independent mechanism (P < 0.05). These effects were reversed when MKK6CA cells were cotransfected with p38gamma-specific siRNA. To determine whether the p38gamma isoform is involved in the regulation of contraction-stimulated glucose uptake in adult skeletal muscle, the tibialis anterior muscles of mice were injected with pCAGGS or wild-type p38gamma (p38gammaWT) followed by intramuscular electroporation. Basal and contraction-stimulated 2-DG uptake in vivo was determined 14 days later. Overexpression of p38gammaWT resulted in higher basal rates of glucose uptake compared with pCAGGS (P < 0.05). Muscles overexpressing p38gammaWT showed a trend for lower in situ contraction-mediated glucose uptake (P = 0.08) and significantly lower total GLUT4 levels (P < 0.05). These data suggest that p38gamma increases basal glucose uptake and decreases DNP- and contraction-stimulated glucose uptake, partially by affecting levels of glucose transporter expression in skeletal muscle. These findings are consistent with the hypothesis that activation of stress kinases such as p38 are negative regulators of stimulated glucose uptake in peripheral tissues.

Oat consumption does not affect resting casual and ambulatory 24-h arterial blood pressure in men with high-normal blood pressure to stage I hypertension.

The results of epidemiologic studies suggest that increased intake of dietary fiber is associated with lower levels of arterial blood pressure (BP). However, there is little information available addressing the possibility that increased oat consumption may reduce arterial BP in individuals with elevated arterial BP. To test this hypothesis, middle-aged and older men (n = 36; body mass index, 25-35 kg/m(2); aged 50-75 y) with elevated BP (systolic BP 130-159 mmHg and/or diastolic BP 85-99 mmHg) were randomly assigned to consume an additional 14 g/d of dietary fiber in the form of oat (5.5 g beta-glucan, n = 18) or wheat cereals (no beta-glucan, n = 18) for 12 wk. Casual resting arterial BP was measured at baseline and after 4, 8 and 12 wk of intervention. The 24-h ambulatory arterial BP was measured at baseline and after 12 wk of intervention. There were no differences in casual resting or 24-h ambulatory BP at baseline in the two groups. Casual systolic BP (SBP) did not change as a result of the 12-wk intervention in the oat (138 plus minus 2 vs. 135 plus minus 3 mmHg) or wheat (142 plus minus 2 vs. 140 plus minus 3 mmHg) groups, respectively (all P > 0.05). Casual diastolic BP (DBP) also did not change in the oat (89 plus minus 2 vs. 88 plus minus 2 mmHg) or wheat (90 plus minus 2 vs. 91 plus minus 2 mmHg) group during this period (all P > 0.05). Further, 24-h, daytime and nighttime SBP and DBP did not decrease with the intervention. Therefore, the results of the present study suggest that any cardioprotective benefit of regular oat consumption may not be conferred via an arterial BP-lowering effect.

Behavioral, metabolic, and molecular correlates of lower insulin sensitivity in Mexican-Americans.

We determined whether lower insulin sensitivity persists in young, nonobese, nondiabetic Mexican-American [MA; n = 13, 27.0 +/- 2.0 yr, body mass index (BMI) 23.0 +/- 0.7] compared with non-Hispanic white (NHW; n = 13, 24.8 +/- 1.5 yr, BMI 22.8 +/- 0.6) males and females after accounting for cardiorespiratory fitness (maximal O(2) uptake), abdominal fat distribution (computed tomography scans), dietary intake (4-day records), and skeletal muscle insulin-signaling protein abundance from muscle biopsies (Western blot analysis). MA were significantly less insulin sensitive compared with their NHW counterparts when estimated by homeostatic model assessment of insulin resistance (MA: 1.53 +/- 0.22 vs. NHW: 0.87 +/- 0.16, P < 0.05) and the revised quantitative insulin sensitivity check index (MA: 0.45 +/- 0.08 vs. NHW: 0.58 +/- 0.19, P = 0.05). However, skeletal muscle protein abundance of insulin receptor-beta (IRbeta), phosphatidylinositol 3-kinase p85 subunit, Akt1, Akt2, and GLUT4 were not significantly different. Differences in indexes of insulin sensitivity lost significance after percent dietary intake of palmitic acid, palmitoleic acid, and skeletal muscle protein abundance of IRbeta were accounted for. We conclude that differences in insulin sensitivity between nonobese, nondiabetic MA and NHW persist after effects of chronic and acute exercise and total and abdominal fat distribution are accounted for. These differences may be mediated, in part, by dietary fat intake.