Rapid inhibition of acetyl-CoA carboxylase (ACC) activity in rat liver in response to 6 h starvation and rapid re-activation in response to 2-6 h of re-feeding chow were shown to be due to changes in the expressed activity of existing enzyme. Decreases and increases in ACC concentration occurred at later stages of the transitions, i.e. 6-48 h starvation and 8-24 h re-feeding respectively. The decrease in expressed activity of ACC was due primarily to changes in its phosphorylation state, demonstrated by a significantly decreased Vmax. and significantly increased Ka for citrate of enzyme purified by avidin-Sepharose chromatography from 6 h- or 48 h-starved rats. These effects were totally reversed within 2-4 h of chow re-feeding. Changes in the activity of purified ACC closely correlated with reciprocal changes in the activity of AMP-activated protein kinase (AMP-PK) over the fed to starved to re-fed transition. Increases in the activity ratio of cyclic-AMP-dependent protein kinase in response to starvation lagged behind the increase in AMP-PK and the decrease in ACC activity. Changes in AMP-PK and ACC activities of rat liver closely correlated with changes in plasma insulin concentration in response to time courses of starvation and re-feeding.
Truncated forms of glucagon-like peptide-1 (tGLP-1) are potent endogenous stimuli of insulin secretion from pancreatic beta cells and have powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the tGLP-1 receptor (R) that are required for its efficient coupling to the adenylyl cyclase (AC) system since it is well established that cAMP is the primary second messenger activated by tGLP-1. The predicted third intracellular loop (IC3) of the rat tGLP-1R was systemically scanned using a mutagenic based strategy. The resulting receptor mutants were expressed in COS-7 cells and examined for cAMP formation in response to tGLP-1 stimulation (10nM) and [125I] tGLP-1(7-36) amide binding. A single block deletion (IC3-1) within the N-terminal region of IC3 (K334-L335-K336) resulted in a dramatic reduction in the cAMP response to tGLP-1 (7.1 +/- 1.4% of the wild type (wt) tGLP-1R response, n = 3, p < or = 0.01), while displaying comparable levels of expression, (expressed as the %Bmax of the wt-tGLP-1R (101 +/- 13%, n = 3, p > or = 0.05). This receptor mutation was further analyzed by stable expression in CHO-K1 cells. In agreement with the COS model, IC3-1 displayed comparable levels of receptor expression (97 +/- 16% Bmax of wt tGLP-1R, n = 3, p > or = 0.05) and affinity for tGLP-1(Kd of 460 +/- 15pM vs. 450 +/- 12pM wt tGLP-1R, n = 3, p > or = 0.05), but was unable to effectively stimulate cAMP production (7.7 +/- 0.4% of wt tGLP-1R, n = 3, p < or = 0.01) in response to tGLP-1 (10nM), No other mutation examined within the IC3 domain displayed a lack of correlation between binding activity and cAMP accumulation. Further analysis of the K334-L335-K336 sequence by substitution analysis revealed that a K334 to A substitution was the only modification to result in a striking attenuation of the cAMP response (28 +/- 1.9% of wt tGLP-1, n = 3, p < or = 0.01). These results strongly suggest that within the IC3 domain the N-terminal KLK sequence or a portion thereof (specifically K-334) is required for the efficient coupling of the tGLP-1 receptor to the AC system.
Protein phosphorylation by cyclic AMP-dependent protein kinase (CAMP-PK) is an important mechanism regulating the flux through many metabolic pathways I 11. In the lactating rat mammary gland fatty acid synthesis is acutely regulated according to the nutritional status of the rat and this is mediated at least in part by the reversible phosphorylation and inactivation of acetyl-CoA carboxylase (ACC) 121. Although thecatalytic (C) subunit of CAMP-PK purified from bovine heart and rabbit skeletal muscle can phosphorylate and inactivate purified mammary ACC in virro 131, increased cAMP concentrations in mammary tissue in response to B-agonists and phosphodiesterase inhibitors do not cause phosphorylation and inactivation of ACC nor inhibition of fatty acid synthesis 141.We believe this lack of physiological effect of cAMP may be partly explained by the fact that the C-subunit of CAMP-PK In lactating mammary gland appears to be a different isoenzyme from that in heart, with a lower affinity for ACC. In most tissues the inactive, tetrameric CAMP-PK holoenzyme (RzCz) is present as two isoforms termed type 1 and I1 according to their order of elution from DEAE cellulose and reportedly differing only in the structure of the regulatory subunit 151. The C-subunit of CAMP-PK from all sources was thought to be invariant based largely on molecular weight and tryptic peptide similarities 151. However, there is increasing evidence for multiple isoenzyme forms of C-subunit and we have found striking differences in the properhes of C-subunits purified from rat heart and lactating rat mammary glandThe purification method was a rapid modification of that in 161. Post-100,000g supernatants were batch absorbed onto DEAE Sepharose and the C-subunit of CAMP-PK was batch eluted, in the same buffer containing lOuM CAMP, directly onto a phosphocellulose column. This column was eluted with an increasing, linear gradient of potassium phosphate. The C-subunits were indistinguishable in terms of apparent molecular weight as estimated by PAGE (Mr = 41,000 Da) and by Superose 12 gel filtration on FPLC Mr = 45,000 Da). The C-subunit from rat heart was considerably more sensitive to the specific peptide inhibitor of CAMP-PK (Sigma Chemical Co., Poole, Dorset) with sequence 'ITYADFIASGRTGRRNAIHD based on the active fragment of Walsh inhibitor 171. Heart CAMP-PK had an ID50 = 0.35 nglml compared to 2.85 ng/ml for mammary gland CAMP-PK. lable 1. Substrate smcificitv of C-subunits of cAMl'-PK from rat heart and lactatine m a m m w gland, C-subunit of CAMP-PK was purified from rat heart and lactating mammary gland, and initial rates of phosphorylation assayed. Results show initial velocities measured at the indicated subsbate concentrations and are expressed relative to the rate of phosphorylation of histone (type WAS).CAMP-PK C-subunit activity (relative to histone = 100%) Mammary gland Heart Histone (0.80 mg/ml) 100 100 ATP-cihate lyase (0.24 mg/ml) 50 7 Glycogen synthase (0.17 mg/ml) 130 110 ACC (0.48 mg/ml) < I 17 Phosphorylase kinase (0.67 mg/ml) ...
Acetyl-CoA carboxylase (ACC) catalyses the first committed step in the synthesis of fatty acids. While this enzyme can be allosterically activated by citrate and inhibited by fatty acyl-CoA? its major form of hormonal regulation is achieved by reversible phosphorylation and inactivation [ 11. Although CAMP-dependent pmtein kinase (CAMP-PK) from bovine heart can phosphorylate and inactivate ACC in vitro [2], there is now much evidence to suggest that the protein k i n a responsible for the phosphorylation and inactivation of ACC in vivo is the AMP-activated protein kinase (AMP-PK) originally identified in rat liver [3]. For example, in hepatocytes [4] and adipcytes [5] it is serine-79 of ACC (the site phosphorylated by AMP-PK in vitro) and not serine-77 (the site phosphorylated by CAMP-PK) that contains phosphate. The extent of ACC Vmax inhibition in vivo can only be matched in vitm by phosphorylation of ACC by AMP-PK [2]. While AMP-PK is activated allosterically by micromolar concentrations of 5'-AMP it is also phosphorylated and activated by a distinct lrinase kinase' activity in a reachon stimulated in vitro by nanomolar palmitoyl-CoA [3]. It would appear that CAMP-PK is not this kinase kinax, but it has been proposed that CAMP-PK is important somewhere in this cascade [5].In rat liver in vivo, ACC is inactivated and reactivated in parallel with changes in fatty acid synthesis in response to time courses of starvation and refeeding, respectively. We have observed that there are parallel reciprocal changes in the activity of AMP-PK ova these time courses that more closely correlate with changes in plasma insulin concatration than with changes in CAMP-PK activity [6]. A tissue in which fatty acid synthesis is acutely regulated and which is highly insulin sensitive and responsive is the lactating rat mammary gland. It has previously been shown that a 98% inhibition of mammary gland fatty acid synthesis in response to 24h starvation, is accompanied by a 75% decrease in the Vmax of ACC as a result of an increase in its phosphate mtent [7]. These effects were reversed by refeeding. We have identified, purified and characterised AMP-PK from lactating rat m a m~ gland and have found no major differences in properties between it and AMP-PK from rat liver although the specific activity at any stage of purification always appears to be lower for the mammary enzyme. Using the synthetic peptide substrate HMRSAMSGLHLVKRR (based on the sequence surrounding the phosphorylation sites on ACC, but specific for AMP-PK because serine-77 is replaced with alanine), we have monitored changes in AMP-PK activity in response to starvation and refeeding in the lactating rat mammary gland. Table 1 confirms previous reports (7, 81 that there was no change in ACC activity during the fist 6h starvation, but that a significant decrease (54%) in Vmax of the purified enzyme had occurred by 24h starvation and this was readily reversed by 3h refeeding. The activity of AMP-PK changed reciprocally with changes in ACC Vmax exhibiting an 88% increase ...
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