Rekha Patel

Age-related differences in the dose–response relationship of muscle protein synthesis to resistance exercise in young and old men

We investigated how myofibrillar protein synthesis (MPS) and muscle anabolic signalling were affected by resistance exercise at 20–90% of 1 repetition maximum (1 RM) in two groups (25 each) of post‐absorptive, healthy, young (24 ± 6 years) and old (70 ± 5 years) men with identical body mass indices (24 ± 2 kg m−2). We hypothesized that, in response to exercise, anabolic signalling molecule phosphorylation and MPS would be modified in a dose‐dependant fashion, but to a lesser extent in older men. Vastus lateralis muscle was sampled before, immediately after, and 1, 2 and 4 h post‐exercise. MPS was measured by incorporation of [1,2‐13C] leucine (gas chromatography–combustion–mass spectrometry using plasma [1,2‐13C]α‐ketoisocaparoate as surrogate precursor); the phosphorylation of p70 ribosomal S6 kinase (p70s6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was measured using Western analysis with anti‐phosphoantibodies. In each group, there was a sigmoidal dose–response relationship between MPS at 1–2 h post‐exercise and exercise intensity, which was blunted (P < 0.05) in the older men. At all intensities, MPS fell in both groups to near‐basal values by 2–4 h post‐exercise. The phosphorylation of p70s6K and 4EBP1 at 60–90% 1 RM was blunted in older men. At 1 h post‐exercise at 60–90% 1 RM, p70s6K phosphorylation predicted the rate of MPS at 1–2 h post‐exercise in the young but not in the old. The results suggest that in the post‐absorptive state: (i) MPS is dose dependant on intensity rising to a plateau at 60–90% 1 RM; (ii) older men show anabolic resistance of signalling and MPS to resistance exercise.

Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle.

Resistance (RE) and endurance (EE) exercise stimulate mixed skeletal muscle protein synthesis. The phenotypes induced by RE (myofibrillar protein accretion) and EE (mitochondrial expansion) training must result from differential stimulation of myofibrillar and mitochondrial protein synthesis. We measured the synthetic rates of myofibrillar and mitochondrial proteins and the activation of signalling proteins (Akt–mTOR–p70S6K) at rest and after an acute bout of RE or EE in the untrained state and after 10 weeks of RE or EE training in young healthy men. While untrained, RE stimulated both myofibrillar and mitochondrial protein synthesis, 67% and 69% (P < 0.02), respectively. After training, only myofibrillar protein synthesis increased with RE (36%, P= 0.05). EE stimulated mitochondrial protein synthesis in both the untrained, 154%, and trained, 105% (both P < 0.05), but not myofibrillar protein synthesis. Acute RE and EE increased the phosphorylation of proteins in the Akt–mTOR–p70S6K pathway with comparatively minor differences between two exercise stimuli. Phosphorylation of Akt–mTOR–p70S6K proteins was increased after 10 weeks of RE training but not by EE training. Chronic RE or EE training modifies the protein synthetic response of functional protein fractions, with a shift toward exercise phenotype‐specific responses, without an obvious explanatory change in the phosphorylation of regulatory signalling pathway proteins.

Blunting of insulin inhibition of proteolysis in legs of older subjects may contribute to age-related sarcopenia

BACKGROUND Reduced postprandial muscle proteolysis is mainly due to increased insulin availability. Whether rates of proteolysis in response to low physiologic doses of insulin are affected by aging is unknown. OBJECTIVES We tested the hypothesis that suppression of leg protein breakdown (LPB) by insulin is blunted in older subjects, together with blunted activation of Akt-protein kinase B (PKB). DESIGN Groups of 8 young [mean (+/-SD) age: 24.5 +/- 1.8 y] and older (65.0 +/- 1.3 y) participants were studied during euglycemic (5 mmol/L), isoaminoacidemic (blood leucine approximately 120 micromol/L) clamp procedures at plasma insulin concentrations of approximately 5 and approximately 15 microIU/mL for 1.5 h. Leg amino acid balance, whole-leg protein turnover (as dilution of amino acid tracers), and muscle protein synthesis were measured with D(5)-phenylalanine and [1,2-(13)C(2)]leucine. The kinase activity of muscle Akt-PKB and the extent of phosphorylation of signaling proteins associated with the mTOR (mammalian target of rapamycin) pathway were measured before and after the clamp procedures. RESULTS Basal LPB rates were not different between groups (66 +/- 11 compared with 51 +/- 10 nmol leucine x 100 mL leg(-1) x min(-1) and 30 +/- 5 compared with 24 +/- 4 nmol phenylalanine x 100 mL leg(-1) x min(-1) in young and older groups, respectively). However, although insulin at approximately 15 microIU/mL lowered LPB by 47% in the young subjects (P < 0.05) and abolished the negative leg amino acid balance, this caused only a 12% fall (P > 0.05) in the older group. Akt-PKB activity mirrored decreases in LPB. No differences were seen in muscle protein synthesis or associated anabolic signaling phosphoproteins. CONCLUSIONS At moderate availability, the effect of insulin on LPB is diminished in older human beings, and this effect may be mediated through blunted Akt-PKB activation.

Mechanism of Exocrine Pancreatic Insufficiency in Streptozotocin-Induced Type 1 Diabetes Mellitus

Abstract:  Diabetes mellitus (DM) is a major health problem at present affecting about 180 million people worldwide. DM is associated with many metabolic abnormalities in the body including the indigestion of carbohydrates leading to malnutrition and weight loss. In this article we investigate the cellular and molecular mechanisms of exocrine pancreatic insufficiency in streptozotocin (STZ, 60 mg kg−1, i.p.)‐induced DM in male rats compared to healthy age‐matched controls. Either electrical field stimulation (EFS) or cholecystokinin octapeptide (CCK‐8, 10−8 M) can elicit large and significant (P < 0.05) increases in amylase output from pancreatic segments compared to basal secretion. Insulin (10−6 M) alone has no significant effect on amylase output compared to basal but it enhanced the secretory responses to either EFS or CCK‐8. When rats were rendered diabetic with STZ, either EFS or CCK‐8‐evoked amylase output was significantly (P < 0.01) decreased compared to the responses obtained with either EFS or CCK‐8 alone in healthy age‐matched control pancreas. In addition, CCK‐8 can elicit large dose‐dependent release of amylase in age‐matched control and diabetic acinar cells with significantly (P < 0.05) reduced responses in diabetic acinar cells. CCK‐8 evoked a large rapid increase in peak cytosolic free calcium concentration ([Ca2+]c) followed by a decrease to a plateau phase in age‐matched control fura‐2‐loaded pancreatic acinar cells. These responses were significantly (P < 0.05) decreased in STZ‐induced diabetic acinar cells. In the presence of 10−6 M insulin, CCK‐8 evoked a much larger increase in the Ca2+ transient compared to the response obtained with CCK‐8 alone. These effects were significantly (P < 0.01) inhibited in STZ‐induced diabetic acinar cells. Similarly, in zero extracellular Ca2+[Ca2+]ĉ, the CCK‐8‐evoked [Ca2+]c was significantly (P < 0.05) reduced in both diabetic and age‐matched control acinar cells, but with more pronounced reduction in diabetic acinar cells. CCKA receptor mRNA levels remained unchanged in diabetic rat acinar cells compared to age‐matched healthy control. In contrast, amylase mRNA was significantly (P < 0.05) reduced in diabetic acinar cells compared to control. The results indicate that reduced amylase secretion in response to either EFS or CCK‐8 in the diabetic pancreas may be due to reduced [Ca2+]c and gene expression for amylase and not to the gene expression of CCKA receptor in pancreatic acinar cells.

Antiangiogenic effects and transcriptional regulation of pigment epithelium-derived factor in diabetic retinopathy

The effects of the antiangiogenic cytokine PEDF on key steps in retinal angiogenesis, specifically endothelial cell proliferation and vascular tubule formation, and the regulation of PEDF expression in retinal capillary endothelial cells were evaluated. HUVECs were co-cultured with fibroblasts to construct a model of angiogenesis using the Angiokit assay, and image analysis software was used to measure the effects of PEDF and VEGF on vascular tubule formation. Quantitative real-time PCR analysis was used to determine the expression of PEDF in microvascular endothelial cells exposed to glucose 20 mM, insulin 100 nM and VEGF 10 ng/ml. PEDF inhibited endothelial cell proliferation and significantly decreased the number of tubules (629+93 AU vs 311+31, p=0.001), number of branching points (145+19 AU vs 46+5, p=0.03) and total tubule length (4848+748 AU vs 11,172+2353, p=0.001). In bovine retinal capillary endothelial cells (BRCECs), PEDF mRNA and protein expression was suppressed by insulin (22%) in a rapamycin-sensitive manner; wortmannin had no effect. PEDF mRNA expression was also significantly reduced in the presence of high glucose (23%) and VEGF (25%). In conclusion, PEDF inhibits key steps in the angiogenic response of BRCECs, including endothelial cell proliferation and vascular tubule formation. Gene expression of PEDF is negatively regulated by glucose, insulin (via an mTOR-dependent pathway) and VEGF.

Signaling Proteins Associated with Diabetic‐Induced Exocrine Pancreatic Insufficiency in Rats

Abstract:  Diabetes mellitus (DM) is associated with pancreatic atrophy and compromised digestion of carbohydrates as a result of exocrine pancreatic insufficiency and lower alpha‐amylase synthesis and secretion. The reduced production of digestive enzymes is likely to be caused by deregulated protein metabolism. The relative concentrations and phosphorylation of signaling proteins associated with protein translation, such as PKB, p70S6K1, 4E‐BP1, ERK1/2, and also some of those implicated in protein breakdown, such as ubiquitin and NF‐κB, in the pancreas of streptozotocin (STZ)‐induced type I diabetic pancreas were measured using Western blotting. There were significant decreases in the levels of total PKB, p70S6K, 4E‐BP1, ERK1/2, and NF‐κB in the diabetic pancreas compared to control. In contrast, the phosphorylation of p70S6K1, 4E‐BP1, ERK1/2, and protein ubiquitination increased significantly compared to controls. Together, these results indicate that STZ‐induced DM leads to reduced levels of enzymes mediating protein synthesis while their phosphorylation is actually increased, perhaps in an attempt to maintain protein homeostasis, which is further compromised by heightened ubiquitin‐dependent protein breakdown. It is likely that these factors are responsible for pancreatic atrophy, enzyme synthesis, and net protein loss in DM.

Effect of Insulin on Acetylcholine‐Evoked Amylase Release and Calcium Mobilization in Streptozotocin‐Induced Diabetic Rat Pancreatic Acinar Cells

Abstract:  This article investigated the effect of acetylcholine (ACh) on amylase secretion and cellular calcium homeostasis [Ca2+]i in streptozotocin (STZ; 60 mg kg−1, intraperitoneally)‐induced diabetic rats compared to age‐matched controls in an attempt to understand the cellular mechanism of exocrine pancreatic insufficiency. ACh‐evoked marked dose‐dependent increases in amylase release from isolated pancreatic acini and acinar cells in healthy control rats. In diabetic acini and acinar cells, the ACh‐evoked amylase release was significantly (P < 0.05) reduced compared to healthy acini and acinar cells. Insulin (10−6M) stimulated amylase release in both control and diabetic acini and acinar cells but with a much reduced effect in diabetic tissues. Combining insulin with ACh had no significant effect on amylase release compared to the effect of ACh alone. In fura‐2 loaded pancreatic acinar cells of normal rats, ACh (10−5M) evoked a large initial rise (peak) in [Ca2+]i followed by a decline into a plateau phase. This effect of ACh was significantly (P < 0.05) reduced in fura‐2 loaded diabetic acinar cells. In control cells, insulin had no significant effect on either basal or ACh evoked [Ca2+]i compared to the effect of ACh alone. In contrast, in diabetic acinar cells, insulin significantly (P < 0.05) attenuated the effect of ACh. In a normally free extracellular Ca2+ medium [Ca2+]o containing 1 mM EGTA, the ACh‐evoked [Ca2+]i in normal healthy fura‐2 loaded acini was similar to the response obtained with ACh in fura‐2 loaded diabetic acini. Together, the results indicated that exocrine pancreatic insufficiency is associated with decreased [Ca2+]i due to less Ca2+ released from internal stores and less Ca2+ entering the cell from the extracellular medium.