ABSTRACT
Metformin is known to improve insulin sensitivity in part via a rise in AMP-activated protein kinase (AMPK) activity and alterations in muscle metabolism. However, a full understanding of how metformin alters AMPK-α(1) vs. AMPK-α(2) activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK-α(1) or AMPK-α(2) protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK-α(2), metformin preferentially activated AMPK-α(1) in a dose- and time-dependent manner. Metformin increased (P < 0.05) glucose uptake and plasma membrane (PM) Glut4 in a dose- and time-dependent manner. Metformin significantly reduced palmitate uptake (P < 0.05) and oxidation (P < 0.05), and this was accompanied by a similar decrease (P < 0.05) in PM CD36 content but with no change in acetyl-CoA carboxylase (ACC) phosphorylation (P > 0.05). AICAR and metformin similarly increased (P < 0.05) nuclear silent mating-type information regulator 2 homolog 1 (SIRT1) activity. Downregulation of AMPK-α(1) completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK-α(2) had no effect on metformin-induced glucose uptake, palmitate uptake, and oxidation. The increase in SIRT1 activity induced by metformin was not affected by downregulation of either AMPK-α(1) or AMPK-α(2). Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK-α(1), and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme.
Subject(s)
Fatty Acids/metabolism , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Muscle, Skeletal/drug effects , Protein Kinases/metabolism , AMP-Activated Protein Kinase Kinases , Acetyl-CoA Carboxylase/analysis , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , CD36 Antigens/analysis , Cell Line , Down-Regulation , Glucose/metabolism , Glucose Transporter Type 4/metabolism , Insulin/pharmacology , Muscle, Skeletal/enzymology , Palmitates/metabolism , Protein Kinases/genetics , RNA Interference , Rats , Ribonucleotides/pharmacology , Sirtuin 1/analysisABSTRACT
Context: Total pancreatectomy and intrahepatic islet autotransplantation (TP/IAT) is performed to alleviate severe abdominal pain, avoid narcotic use, maintain islet function, and avoid diabetes in patients with chronic pancreatitis. However, many TP/IAT recipients complain of postprandial hypoglycemia. Objective: This study was designed to discover the mechanisms of this problem. Design: Participants consumed a triple-isotope mixed meal. Setting: This study was performed in a hospital research unit. Participants: We studied 10 TP/IAT recipients and 10 age- and body mass index-matched control subjects. Seven of 10 recipients had a history of postprandial hypoglycemia. Interventions: Participants were given a [1-13C]-labeled mixed meal and two tracer infusions ([6,6-2H2]- and [6-3H]-glucose). Main Outcome Measures: Glucose kinetics and concentrations of regulatory hormones were determined. Results: Immediately after the meal, peak glucose was elevated in recipients compared with control subjects [266 ± 20 mg/dL (14.8 ± 1.1 mmol/L) vs 185 ± 13 mg/dL (10.3 ± 0.7 mmol/L); P = 0.01]. However, mean Δ glucose for TP/IAT recipients between minutes 240 and 360 postprandially was significantly lower than for control subjects (P < 0.05); six of the seven recipients with a history of hypoglycemia experienced abnormally low postprandial Δ glucose. Δ Glucagon remained unchanged (minutes 240 to 360; P = 0.58) in TP/IAT recipients despite abnormal decreases in postprandial glucose. Radioisotopic studies revealed that meal appearance, glucose disappearance, and endogenous glucose production in TP/IAT recipients were not different from control subjects. Conclusion: Initially high glucose levels followed by hypoglycemia with an absent glucagon response is a mechanistic sequence that contributes to postprandial hypoglycemia after TP/IAT.
Subject(s)
Blood Glucose/metabolism , Glucagon/blood , Hypoglycemia/blood , Islets of Langerhans Transplantation/methods , Pancreatectomy , Pancreatitis, Chronic/surgery , Adult , Female , Humans , Male , Meals , Middle Aged , Pancreatitis, Chronic/blood , Postprandial Period/physiology , Transplantation, AutologousABSTRACT
Context: Total pancreatectomy followed by intrahepatic islet autotransplantation (TP/IAT) is performed to alleviate severe, unrelenting abdominal pain caused by chronic pancreatitis, to improve quality of life, and to prevent diabetes. Objective: To determine the cause of exercise-induced hypoglycemia that is a common complaint in TP/IAT recipients. Design: Participants completed 1 hour of steady-state exercise. Setting: Hospital research unit. Patients and Other Participants: We studied 14 TP/IAT recipients and 10 age- and body mass index-matched control subjects. Interventions: Peak oxygen uptake (VO2) was determined via a symptom-limited maximal cycle ergometer test. Fasted subjects then returned for a primed [6,6-2H2]-glucose infusion to measure endogenous glucose production while completing 1 hour of bicycle exercise at either 40% or 70% peak VO2. Main Outcome Measures: Blood samples were obtained to measure glucose metabolism and counterregulatory hormones before, during, and after exercise. Results: Although the Borg Rating of Perceived Exertion did not differ between recipients and control subjects, aerobic capacity was significantly higher in controls than in recipients (40.4 ± 2.0 vs 27.2 ± 1.4 mL/kg per minute; P < 0.001). This difference resulted in workload differences between control subjects and recipients to reach steady-state exercise at 40% peak VO2 (P = 0.003). Control subjects significantly increased their endogenous glucose production from 12.0 ± 1.0 to 15.2 ± 1.0 µmol/kg per minute during moderate exercise (P = 0.01). Recipients did not increase endogenous glucose production during moderate exercise (40% peak VO2) but succeeded during heavy exercise, from 10.1 ± 0.4 to 14.8 ± 2.0 µmol/kg per minute (70% peak VO2; P = 0.001). Conclusions: Failure to increase endogenous glucose production during moderate exercise may be a key contributor to the hypoglycemia TP/IAT recipients experience.
Subject(s)
Exercise Tolerance/physiology , Exercise/physiology , Glucose/metabolism , Islets of Langerhans Transplantation/methods , Pancreatectomy/methods , Pancreatitis, Chronic/surgery , Adult , Blood Glucose/analysis , Case-Control Studies , Female , Follow-Up Studies , Heart Rate/physiology , Humans , Male , Oxygen Consumption/physiology , Pancreatitis, Chronic/diagnosis , Risk Assessment , Sampling StudiesABSTRACT
We used intravenous arginine with measurements of insulin, C-peptide, and glucagon to examine ß-cell and α-cell survival and function in a group of 10 chronic pancreatitis recipients 1-8 years after total pancreatectomy and autoislet transplantation. Insulin and C-peptide responses correlated robustly with the number of islets transplanted (correlation coefficients range 0.81-0.91; P < 0.01-0.001). Since a wide range of islets were transplanted, we normalized the insulin and C-peptide responses to the number of islets transplanted in each recipient for comparison with responses in normal subjects. No significant differences were observed in terms of magnitude and timing of hormone release in the two groups. Three recipients had a portion of the autoislets placed within their peritoneal cavities, which appeared to be functioning normally up to 7 years posttransplant. Glucagon responses to arginine were normally timed and normally suppressed by intravenous glucose infusion. These findings indicate that arginine stimulation testing may be a means of assessing the numbers of native islets available in autologous islet transplant candidates and is a means of following posttransplant α- and ß-cell function and survival.
Subject(s)
Arginine/pharmacology , Glucagon-Secreting Cells/cytology , Glucagon-Secreting Cells/physiology , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/physiology , Islets of Langerhans Transplantation , Adult , Female , Glucagon-Secreting Cells/drug effects , Humans , Insulin-Secreting Cells/drug effects , MaleABSTRACT
Protease inhibitors (PIs), such as atazanavir sulfate and ritonavir, are used clinically to prevent the progression of HIV and are known to induce insulin resistance. To determine whether PI-mediated insulin resistance is induced by activation of pro-inflammatory cascades, L6 skeletal muscle cells were treated ±atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir, and ±insulin. Treatment with atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir for 24 or 48 h significantly increased basal glucose uptake (P<0.05) and atazanavir sulfate + ritonavir treatment increased basal glucose uptake significantly more than ritonavir or atazanavir sulfate treatment alone (P<0.05). Atazanavir sulfate + ritonavir treatment for 48 h completely prevented insulin stimulation of glucose uptake (P>0.05). When compared to untreated cells, basal palmitate uptake and oxidation was found to be significantly higher in cells treated with PIs alone or in combination (P<0.05). Prior PI treatment alone or in combination prevented (P>0.05) the insulin-mediated increase in palmitate uptake and the insulin-mediated decrease in palmitate oxidation observed in the control group. Atazanavir sulfate treatment alone or in combination with ritonavir significantly increased JNK1/2 phosphorylation when compared to the control or ritonavir group (P<0.05) and this was accompanied by a rise (P<0.05) in AKT(Ser473) phosphorylation in the basal state. Total JNK1/2 and p38 MAPK protein content and p38 MAPK phosphorylation state were not altered in any of the treatment groups (P>0.05). Our data indicate that, in muscle cells, PIs induce metabolic dysfunction that is not limited to insulin-sensitive metabolism and that is potentially mediated by a rise in JNK1/2 pro-inflammatory signaling.
Subject(s)
HIV Protease Inhibitors/pharmacology , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Muscle Fibers, Skeletal/drug effects , Signal Transduction/drug effects , Animals , Atazanavir Sulfate , Cell Line , Glucose/metabolism , Insulin Resistance , Muscle Fibers, Skeletal/enzymology , Muscle Fibers, Skeletal/metabolism , Oligopeptides/pharmacology , Oxidation-Reduction/drug effects , Palmitates/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Pyridines/pharmacology , Rats , Ritonavir/pharmacology , Time Factors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.
Subject(s)
AMP-Activated Protein Kinases/deficiency , Calcium Signaling , Energy Metabolism , Glucose/metabolism , Muscle Contraction , Muscle, Skeletal/enzymology , Palmitic Acid/metabolism , AMP-Activated Protein Kinases/genetics , Animals , Caffeine/pharmacology , Calcium Signaling/drug effects , Energy Metabolism/drug effects , Energy Metabolism/genetics , Gene Expression Regulation , Hindlimb , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Contraction/genetics , Muscle Strength , Muscle, Skeletal/blood supply , Muscle, Skeletal/drug effects , Oxidation-Reduction , Perfusion , Time Factors , Triglycerides/metabolismABSTRACT
OBJECTIVE: Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat. RESEARCH DESIGN AND METHODS: Elective surgery patients received a bolus of [1-(14)C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured. RESULTS: Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 µmol · kg adipose lipid(-1) · min(-1), P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P < 0.001), and both were greater in women than men (P < 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity. CONCLUSIONS: Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation.
Subject(s)
Adipocytes/metabolism , Fatty Acids, Nonesterified/metabolism , Intra-Abdominal Fat/metabolism , Obesity/metabolism , Subcutaneous Fat/metabolism , Adult , Body Composition/physiology , CD36 Antigens/metabolism , Coenzyme A Ligases/metabolism , Diacylglycerol O-Acyltransferase/metabolism , Fatty Acid Transport Proteins/metabolism , Female , Humans , MaleABSTRACT
A method is described for assembling an evaporation manifold from a cell culture flask, which allows for efficient nitrogen evaporation of hexane from 24- and 96-well plates. The precursor parts are readily available in most research laboratories. The nitrogen evaporation manifold is inexpensive, could possibly be used with other organic solvents, and appears ideal for a small number of samples in a multi-well format.