Roger A. Clegg

Prolactin and the regulation of adipose-tissue metabolism during lactation in rats

Removal of litters from young lactating rats for 24 or 48 h or treatment of lactating rats with bromocriptine increased the rate of fatty acid synthesis and the activities of lipoprotein lipase and fatty acid synthetase in adipose tissue, decreased the lipoprotein lipase and fatty acid synthetase activities of mammary gland and lowered the serum-prolactin concentration. Concurrent injections of prolactin prevented the effects of bromocriptine and 24 h of litter removal on most of these changes in adipose tissue, but did not prevent the effects of 48 h of litter removal. The results suggest that effects of prolactin on adipose-tissue metabolism are dependent on a functional mammary gland. Most of the responses of adipose tissue to litter removal were reduced in older rats.

Chapter 4 – The metabolism of white adipose tissue in vivo and in vitro

This chapter presents the adipose tissue metabolism of white adipose tissue that is derived from the studies in vivo and in vitro . Measurement of the rates of fatty acid synthesis (FAS) and fatty acid esterification using 3 H 2 O in vivo requires minimal stress to the animal. Therefore, it is important to accustom animals to handling and to their surroundings because stress leads to significant changes in circulating catecholamine levels. Lipoprotein lipase (LPL) activities in all cases exceed fatty acid uptake rates, and these uptake rates are higher when measured in vivo than in vitro . The similar rates of FAS measured in vivo and in vitro , coupled with the observation that changes in blood flow can tend to impede rather than facilitate changes in FAS, suggest that the major factors involved in the regulation of FAS in adipose tissue will be the amounts and activities of key lipogenic enzymes and translocases. The ratio of free fatty acids (FFA) to glycerol release suggests considerable re-esterification. Rates of re-esterification are measured in vivo using 3 H 2 O. FFA and glycerol turnover rates are also used to estimate their rates of release from adipose tissue. Stimulation of the sympathetic innervation promotes lipolysis but also causes vasoconstriction which is sufficient to delay the release of FFA and, to a lesser extent, glycerol from the tissue. Accumulation of FFA in fat cells in vitro can lead to an inhibition of lipolysis but it is doubtful if sufficiently high concentrations occur in non-pathological conditions in vivo .

Salt-resistant (hepatic) lipase. Evidence for its presence in bovine liver and adrenal cortex

Alkaline lipolytic activities in bovine liver and adrenal cortex have been investigated; each tissue has a salt-resistant, hepatic-type lipase activity of which we describe a partial purification. Properties of the partially purified enzymes have been compared directly with those of authentic hepatic lipase prepared from rat liver. Furthermore, a similar activity has been detected in bovine post-heparin plasma. These findings contrast with a previous report that bovine post-heparin plasma and liver extracts lack hepatic salt-resistant lipase.

Evidence for a distinct H7-resistant form of protein kinase C in rat anterior pituitary gland.

Inhibition of phorbol 12,13‐dibutyrate‐induced protein kinase C (PKC) activity from rat midbrain, anterior pituitary and a number of other tissues, as well as COS 7 cells, was studied in vitro. In anterior pituitary, Ca2+‐independent activity was notably resistant to H7 but sensitive to staurosporine and Ro 31‐8220. All Ca2+‐dependent activity was sensitive to these three inhibitors. Mezerein and 1,2‐dioctanoyl‐sn‐glycerol also activated this H7‐insensitive PKC from anterior pituitary. The distribution of this activity, prominently expressed in pituitary and perhaps also lung, and its characteristic resistance to H7 but not other inhibitors, does not obviously correlate with that of any of the well‐characterised PKCs, and may reflect either a novel or a modified isoform.

Casein secretion in mammary tissue: tonic regulation of basal secretion by protein kinase A.

Despite its quantitative importance in the secretion of lactoproteins, little is known about the triggering and control mechanisms that initiate, regulate and terminate the operation of the basal pathway of lactoprotein secretion throughout the lactation cycle. This study investigated the possible modulation by cAMP-mediated mechanisms, of cellular transit of newly-synthesised caseins and their basal secretion in explants of mammary tissue from lactating rats and rabbits. Enhancement of the rate of secretion of newly-synthesised caseins occurs when mammary explants are challenged in vitro with agents that activate protein kinase A (PKA). Inhibition of PKA slows casein secretion. The PKA-sensitive step(s) in casein secretion is early in the exocytosis pathway but inhibition of PKA does not impair casein maturation. Ultrastructural, immunochemical and biochemical methods locate PKA on membranes of vesicles situated in the Golgi region. Exposure of tissue to a cell-permeant PKA inhibitor results in morphological modification of these vesicular structures. We conclude that PKA mediates tonic positive regulation of the basal secretory pathway for lactoproteins in the mammary epithelial cell.

Cholesterol ester hydrolysis and hormone-sensitive lipase in lactating rat mammary tissue.

Neutral cholesterol esterase activity is expressed in extracts of mammary epithelial cells. The identity of the enzyme catalyzing this hydrolysis was investigated. Anti-hormone-sensitive lipase immunoglobulin elicited the total inhibition of this activity and also immunoprecipitated a single phosphoprotein of Mr 84 kDa from mammary cell extracts previously phosphorylated in vitro with [gamma-32P]ATP and cyclic AMP-dependent protein kinase. It is concluded that mammary cell cholesterol esterase activity results from the presence of hormone-sensitive lipase.

Regulation of adipocyte insulin receptor number and metabolism during late-pregnancy

The regulation of the rate of fatty acid synthesis of rat adipose tissue during late-pregnancy has been investigated. Rats at day 18 of pregnancy were injected over a 2-day period with either prostaglandin F2 alpha (PGF2 alpha), PGF2 alpha plus progesterone, PGF2 alpha plus bromocriptine or carrier solutions, and were then killed on day 20 of pregnancy. Injections of PGF2 alpha resulted in a decreased rate of fatty acid synthesis, a lower serum-insulin concentration, and a reduced number of insulin receptors of adipocytes. Concurrent injections of progesterone, but not of bromocriptine, along with the PGF2 alpha prevented these effects of the latter. The results are consistent with our previous suggestion that the fall in serum progesterone concentration prior to parturition results in a reduction in the number of insulin receptors of adipocytes, which, along with a fall in the serum-insulin concentration, leads to a decrease in the anabolic activity of the tissue.

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca2+ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC α were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C α, β1, δ, ɛ, and ζ/ι were all present in rat midbrain cytosolic extract, PKC α, β1, δ, and ζ/ι were present in spleen cytosol, and PKC α and ζ/ι were present in COS 7 cell cytosol. The predominance of α and β activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca2+ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca2+-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca2+, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca2+ whereas in the presence of Ca2+, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca2+. Maximum values for Ca2+-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca2+-dependent PKC isoform activity in a Ca2+ free environment. The greater potency of mezerein on PKC α was confirmed using PKCα and β further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca2+ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca2+-dependent isoforms in Ca2+ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca2+. The concentration-dependence of DOG was examined on PKC α and PKC β further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC α. However, [3H]PDBu binding data using PKC α purified from several sources gave very different IC50 values when DOG was used as a displacer, and in general these values correlated with the EC50 values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.

Adaptations of adipose tissue metabolism and number of insulin receptors in pregnant sheep

The endocrine control of adipose tissue metabolism during pregnancy in sheep has been investigated. The number of insulin receptors of sheep adipocytes was increased during pregnancy. There was no apparent change in the concentration of serum insulin during pregnancy in sheep while the rise in serum progesterone concentration was smaller and more gradual than in rats. Net lipid deposition in adipocytes occurred during the first 55 days of pregnancy, probably due primarily to increased lipoprotein lipase activity. Net deposition of lipid had ceased by mid-pregnancy while by 125 days of pregnancy, the rate of fatty acid synthesis in adipose tissue was decreased and the serum fatty acid concentration had risen, suggesting the onset of net lipid mobilization in the tissue. Results are compared with those from other studies with rats; it would appear that different mechanisms regulate lipid deposition during pregnancy in sheep and rats.

Adipose tissue metabolism during early pregnancy in the rat: temporal relationships of changes in the metabolic activity, number of insulin receptors, and serum hormone concentrations.

The temporal relationships between changes in rates of fatty acid and acylglycerol glycerol synthesis; the activity of lipoprotein lipase of parametrial adipocytes and their capacity to bind insulin; and the serum concentrations of insulin, progesterone, prolactin, and total lactogenic activity have been examined in rats during the first 15 days of pregnancy. The rate of fatty acid and acylglycerol synthesis showed a transient increase at Days 9 and 12 of pregnancy, whereas there was no change in the activity of lipoprotein lipase activity except for a fall between Days 12 and 15 of pregnancy. The capacity of adipocytes to bind insulin was increased by Day 6 of pregnancy and remained elevated until at least Day 15; no changes in the affinity for insulin were observed. Serum progesterone, insulin, and total lactogenic activity were elevated by Days 3, 9, and 12 of pregnancy, respectively. The results show that progesterone but not placental lactogen could be responsible for the rise in the insulin-binding capacity of rat adipocytes during pregnancy, whereas the fall in lipogenic capacity at about Day 12 of pregnancy coincides with the rise in serum placental lactogen.