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• W2058421805 abstract "Cpefat/fat mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpefatmutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in theCpefat/fat mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly4-Lys5-Arg6] and TRH-[Gly4-Lys5] represent approximately 45% of the total TRH-like immunoreactivity inCpefat/fat mice; they constitute ∼1% in controls. Levels of TRH-[Gly4] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly4-Lys5] and TRH-[Gly4-Lys5-Arg6] to TRH-[Gly4]. When Cpefat/fat mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that theCpefat mutation can affect not only carboxypeptidase activity but also endoproteolysis. BecauseCpefat/fat mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice. Cpefat/fat mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpefatmutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in theCpefat/fat mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly4-Lys5-Arg6] and TRH-[Gly4-Lys5] represent approximately 45% of the total TRH-like immunoreactivity inCpefat/fat mice; they constitute ∼1% in controls. Levels of TRH-[Gly4] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly4-Lys5] and TRH-[Gly4-Lys5-Arg6] to TRH-[Gly4]. When Cpefat/fat mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that theCpefat mutation can affect not only carboxypeptidase activity but also endoproteolysis. BecauseCpefat/fat mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice. Neuropeptides and peptide hormones are first biosynthesized as precursors that must undergo a series of conversions to become biologically active (1Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Google Scholar, 2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Google Scholar). Typically, maturation of these precursors begins with limited proteolysis. Excision of the pro-peptide usually occurs at monobasic, dibasic, or tetrabasic residues where subtilisin-like processing enzymes cleave the precursor on the C-terminal side of these amino acids. The endoproteolysis is usually followed by the sequential removal of the basic amino acid(s) residues by a carboxypeptidase-like enzyme (3Fricker L.D. Ann. Rev. Physiol. 1988; 50: 309-321Google Scholar). In some situations, additional modifications can occur in the form of N-terminal acetylation or pyroglutamate formation, sulfation, and C-terminal amidation (4Eipper B.A. Stoffers D.A. Mains R.E. Ann. Rev. Neurosci. 1992; 15: 57-85Google Scholar). These alterations usually serve to yield a peptide that is both biologically active and resistant to degradation.Further evidence that peptide processing is physiologically relevant has come from a mouse identified at the Jackson Laboratories. This animal was reported to be obese, diabetic, and infertile because of a spontaneous mutation in the fat gene (5Coleman D.L. Eichler E.M. J. Hered. 1990; 81: 424-427Google Scholar). Subsequent studies have shown that the fat/fat mouse has a single point mutation in the carboxypeptidase E (CPE) 1The abbreviations used are: CPE, carboxypeptidase E; CPD, carboxypeptidase D; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; IR, immunoreactivity; PC, prohormone convertase; RIA, radioimmunoassay; T3, triiodothyronine; T4, thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. 1The abbreviations used are: CPE, carboxypeptidase E; CPD, carboxypeptidase D; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; IR, immunoreactivity; PC, prohormone convertase; RIA, radioimmunoassay; T3, triiodothyronine; T4, thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. gene, and hence, this mutation has been termed Cpefat (6Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Google Scholar). A Ser202 to Pro202 transition renders the enzyme catalytically inactive and subject to rapid degradation soon after synthesis. With respect to the diabetes in theCpefat/fat mouse, the mechanism underlying this dysfunction has been partially attributed to a deficiency in converting pro-insulin intermediates to insulin.To date, the mechanisms underlying the obesity in theCpefat/fat mouse are not well understood. The reason for these circumstances is probably because of the multifactorial nature of the regulation of food intake, absorption, and caloric utilization (7Leiter E.H. Kintner J. Flurkey K. Beamer W.G. Naggert J.K. Endocrine. 1999; 10: 57-66Google Scholar, 8Fricker L.D. Leiter E.H. Trends Biochem. Sci. 1999; 24: 390-393Google Scholar). In the latter case, the control of metabolism may be especially important. One hormonal system that plays an important role in the regulation of metabolism is the hypothalamic-pituitary-thyroid axis that is controlled by thyrotropin-releasing hormone (TRH).Hypophysiotrophic pro-TRH is synthesized in the hypothalamic paraventricular nucleus, and it must undergo a number of different processing steps to yield mature TRH (9Nillni E.A. Sevarino K.A. Endocrinol. Rev. 1999; 20: 599-648Google Scholar). Upon stimulation, TRH is released from median eminence nerve terminals into the hypophyseal blood where it is transported to the pituitary to stimulate the biosynthesis and secretion of thyroid-stimulating hormone (TSH; see Ref. 10Reichlin S. Wilson J.D. Foster D.W. Kronenberg H.M. Larsen P.R. Williams Textbook of Endocrinology. 9th Ed. W. B. Saunders Co., Philadelphia, PA1998: 165-248Google Scholar). TSH, in turn, is carried in blood to the thyroid where it stimulates thyroid hormone biosynthesis and release. Besides this role, TRH can also exert some control over the release of prolactin, growth hormone, vasopressin, and insulin, as well as the classic neurotransmitters, norepinephrine and epinephrine. Furthermore, TRH is present in brain regions outside of the hypothalamus where it may serve as a neurotransmitter or neuromodulator.In previous reports, it has been shown that the pro-TRH is cleaved endoproteolytically by two different members of the prohormone convertase (PC) family of enzymes, PC1/3 and PC2 (11Schaner P. Todd R.B. Seidah N.G. Nillni E.A. J. Biol. Chem. 1997; 272: 19958-19968Google Scholar). In this scheme, PC1/3 appears to be primarily responsible for most of the major cleavage events. By contrast, although PC2 can perform many of these same conversions, this enzyme is required for the formation of the pEH24 peptide, and it is specifically involved in the processing of prepro-TRH178–199 to generate the pFQ7 and pSE14 peptides (11Schaner P. Todd R.B. Seidah N.G. Nillni E.A. J. Biol. Chem. 1997; 272: 19958-19968Google Scholar, 12Nillni E.A. Aird F. Seidah N.G. Todd R.B. Koenig J.I. Endocrinology. 2001; 142: 896-906Google Scholar). Because PC1/3 and PC2 seem to cleave the pro-TRH on the C-terminal side of dibasic residues, these basic amino acids must be removed from the pro-TRH intermediates for the TRH to be active biologically. An enzyme that has been hypothesized to produce these conversions is CPE. Because theCpefat/fat mouse has a mutation in this enzyme, this animal should allow us to examine this hypothesis and determine whether this mutation exerts any effects on hypothalamic-pituitary-thyroid function.DISCUSSIONCPE is an exopeptidase that is responsible for removing basic amino acids from the C-terminal of proteins and peptides (3Fricker L.D. Ann. Rev. Physiol. 1988; 50: 309-321Google Scholar). A point mutation in the CPE gene (e.g. Ser202to Pro202 transition) is sufficient to reduce the efficiency of endoproteolytic cleavage of the pro-TRH and its high molecular mass products in Cpefat/fathypothalamus. These data indicate that mutation of this gene can exert effects upstream of its normal processing activity. In rats, pro-TRH is processed by PC 1/3 and 2 (11Schaner P. Todd R.B. Seidah N.G. Nillni E.A. J. Biol. Chem. 1997; 272: 19958-19968Google Scholar, 12Nillni E.A. Aird F. Seidah N.G. Todd R.B. Koenig J.I. Endocrinology. 2001; 142: 896-906Google Scholar). Both endoproteases are synthesized as pro-enzymes and must be processed to attain full activity (1Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Google Scholar, 2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Google Scholar). It has been proposed that CPE may participate in the full activation of these convertases through removal of C-terminal basic amino acids or by inactivation of endogenous inhibitors (27Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Google Scholar, 28Jutras I. Seidah N.G. Reudelhuber T.L. Brechler V. J. Biol. Chem. 1997; 272: 15184-15188Google Scholar, 29Fricker L.D. McKinzie A.A. Sun J. Curran E. Qian Y. Yan L. Patterson S.D. Courchesne P.L. Richards B. Levin N. Mzhavia N. Devi L.A. Douglass J. J. Neurosci. 2000; 20: 639-648Google Scholar). Recently, it has been reported that protein levels and enzymatic activities of both enzymes are altered in the brains of the Cpefat/fat mice (30Berman Y. Mzhavia N. Polonskaia A. Devi L.A. J. Biol. Chem. 2001; 276: 1466-1473Google Scholar). As a consequence, levels of pro-dynorphin and its high molecular mass intermediate (e.g. dynorphin A-17) are increased (30Berman Y. Mzhavia N. Polonskaia A. Devi L.A. J. Biol. Chem. 2001; 276: 1466-1473Google Scholar,31Fricker L.D. Berman Y.L. Leiter E.H. Devi L.A. J. Biol. Chem. 1996; 271: 30619-30624Google Scholar). Additionally, concentrations of pro-insulin in pancreas (6Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Google Scholar) and pro-opiomelanocortin in pituitary are enhanced (29Fricker L.D. McKinzie A.A. Sun J. Curran E. Qian Y. Yan L. Patterson S.D. Courchesne P.L. Richards B. Levin N. Mzhavia N. Devi L.A. Douglass J. J. Neurosci. 2000; 20: 639-648Google Scholar, 31Fricker L.D. Berman Y.L. Leiter E.H. Devi L.A. J. Biol. Chem. 1996; 271: 30619-30624Google Scholar, 32Shen F.-S. Loh Y.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5314-5319Google Scholar). Despite these findings, impaired endoproteolysis of pro-peptides is not necessarily a common feature of the CPE mutation, because the levels of pro-cholecystokinin in brain are unchanged (33Cain B.M. Wang W. Beinfeld M.C. Endocrinology. 1997; 138: 4034-4037Google Scholar, 34Lacourse K.A. Friis-Hansen L. Samuelson L.C. Rehfeld J.F. FEBS Lett. 1998; 436: 61-66Google Scholar), whereas those in intestine are reported to be either enhanced (34Lacourse K.A. Friis-Hansen L. Samuelson L.C. Rehfeld J.F. FEBS Lett. 1998; 436: 61-66Google Scholar) or unaltered (33Cain B.M. Wang W. Beinfeld M.C. Endocrinology. 1997; 138: 4034-4037Google Scholar) from the WT controls.Besides alterations in TRH levels, concentrations of some high and low molecular mass pro-TRH intermediates were also changed in theCpefat/fat hypothalamus. Although the function of TRH has been studied for many years, only recently have physiological roles been ascribed to some of the other pro-TRH products (9Nillni E.A. Sevarino K.A. Endocrinol. Rev. 1999; 20: 599-648Google Scholar). For instance, although 100-fold less potent than TRH, TRH-Gly4 stimulates gastric acid secretion in a dose-dependent manner (35Tache Y. Stephens Jr., R.L. Ishikawa T. Ann. N. Y. Acad. Sci. 1989; 553: 269-285Google Scholar). Additional peptides that have biological activity include the prepro-TRH160–169 and the prepro-TRH178–199 (36Roussel J.-P. Hollande F. Bulant M. Astier H. Neuroendocrinology. 1991; 54: 559-565Google Scholar, 37Redei E. Hilderbrand H. Aird F. Endocrinology. 1995; 136: 3557-3563Google Scholar). The prepro-TRH160–169 augments TRH-stimulated TSH secretion and potentiates TRH-induced gastric acid secretion when microinjected into the dorsal motor nucleus of the vagus nerve. The prepro-TRH178–199 can serve as a corticotropin-releasing hormone inhibiting factor (37Redei E. Hilderbrand H. Aird F. Endocrinology. 1995; 136: 3557-3563Google Scholar, 38McGivern R.F. Rittenhouse P. Aird F. Van de Kar L.D. Redei E. J. Neurosci. 1997; 17: 4886-4894Google Scholar), and it can stimulate prolactin release from the pituitary (12Nillni E.A. Aird F. Seidah N.G. Todd R.B. Koenig J.I. Endocrinology. 2001; 142: 896-906Google Scholar). Inasmuch as these pro-TRH-derived peptides possess biological activity, and because processing of the pro-TRH intermediates are affected by theCPE mutation in mice, it may be expected that some of these functions will be abnormal in the Cpefat/fatmouse.In the present study, hypothalami fromCpefat/fat mice were found to contain high levels of the low molecular mass intermediates, TRH-[Gly4-Lys5-Arg6], and TRH-[Gly4-Lys5]. These data indicate that carboxypeptidase activity is deficient in vivo and that CPE is primarily responsible for this activity in TRH neurons. A role for CPE in TRH processing is strengthened further by the in vitro conversion of TRH-[Gly4-Lys5-Arg6] and TRH-[Gly4-Lys5] to TRH-[Gly4] and by the co-localization of CPE-like IR in TRH neurons. It should be noted that besides TRH intermediates, an increase in additional C-terminal basic amino acid-extended peptides has been observed for insulin (6Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Google Scholar), cholecystokinin (33Cain B.M. Wang W. Beinfeld M.C. Endocrinology. 1997; 138: 4034-4037Google Scholar, 34Lacourse K.A. Friis-Hansen L. Samuelson L.C. Rehfeld J.F. FEBS Lett. 1998; 436: 61-66Google Scholar), gastrin (39Udupi V. Gomez P. Song L. Varlamov O. Reed J.T. Leiter E.H. Fricker L.D. Greeley Jr., G.H. Endocrinology. 1997; 138: 1959-1963Google Scholar, 40Hansen L.F. Rehfeld J.F. Biochem. Biophys. Res. Commun. 2000; 267: 638-642Google Scholar), and neurotensin in the Cpefat mutant (41Rovere C. Viale A. Nahon J.-L. Kitabgi P. Endocrinology. 1996; 137: 2954-2958Google Scholar).In murine hypothalamus, the Cpefat mutation depresses the levels of fully processed TRH. Additional peptides in brain that are also reduced include dynorphin (30Berman Y. Mzhavia N. Polonskaia A. Devi L.A. J. Biol. Chem. 2001; 276: 1466-1473Google Scholar, 31Fricker L.D. Berman Y.L. Leiter E.H. Devi L.A. J. Biol. Chem. 1996; 271: 30619-30624Google Scholar), cholecystokinin (33Cain B.M. Wang W. Beinfeld M.C. Endocrinology. 1997; 138: 4034-4037Google Scholar), neurotensin (41Rovere C. Viale A. Nahon J.-L. Kitabgi P. Endocrinology. 1996; 137: 2954-2958Google Scholar), and substance P (42Perloff M.D. Kream R.M. Beinfeld M.C. Peptides. 1998; 19: 1115-1117Google Scholar). Besides removal of basic amino acids, amidation may also be affected in theCpefat/fat mouse. For instance, the molar ratio of TRH-[Gly4] to TRH and the percent of this glycine-extended peptide relative to the other TRH-like low molecular mass peptides were depressed in mutant hypothalami. A similar relationship has also been reported for glycine-extended cholecystokinin (34Lacourse K.A. Friis-Hansen L. Samuelson L.C. Rehfeld J.F. FEBS Lett. 1998; 436: 61-66Google Scholar) but not for gastrin (40Hansen L.F. Rehfeld J.F. Biochem. Biophys. Res. Commun. 2000; 267: 638-642Google Scholar) in gut. Together, these data suggest that amidative activity may be influenced in theCpefat/fat mouse.Despite perturbations in pro-TRH processing,Cpefat/fat hypothalamus contains low quantities of TRH. This result suggests either that some residual CPE is present or that an additional carboxypeptidase resides in TRH neurons. One possible candidate is CPD. This enzyme is expressed in many brain regions including the hypothalamic paraventricular nucleus where the hypophyseal TRH neurons are located (25Dong W. Fricker L.D. Day R. Neuroscience. 1999; 89: 1301-1317Google Scholar). Moreover, CPD is co-localized with CPE in many different brain regions. In the present report, we show that hypothalamic TRH neurons contain CPE- and CPD-like IR. Moreover, recombinant CPD can process synthetic TRH-[Gly4-Lys5-Arg6] or TRH-[Gly4-Lys5] in vitro. These findings clearly show that CPD can mimic the actions of CPE. Despite this fact, hypothalamic levels of TRH-[Gly4-Lys5-Arg6] and TRH-[Gly4-Lys5] are augmented inCpefat/fat mice. One reason for the discrepancy between the in vitro and in vivo results may be the differential locations of the enzymes. Our immunocytochemistry results show that CPE-like IR is located in the perikarya and processes, whereas the CPD-like IR is confined to the cell body region of TRH neurons. These findings complement other studies where CPE has been localized primarily to mature secretory granules (43Fricker L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3886-3890Google Scholar), whereas CPD resides in the trans-Golgi network and immature secretory granules (44Varlamov O. Eng F.J. Novikova E.G. Fricker L.D. J. Biol. Chem. 1999; 274: 14759-14767Google Scholar). Because pro-TRH processing begins in the Golgi and continues in the secretory granule (16Nillni E.A. Sevarino K.A. Jackson I.M.D. Endocrinology. 1993; 132: 1271-1277Google Scholar, 45Perez de la Cruz I. Nillni E.A. J. Biol. Chem. 1996; 271: 22736-22745Google Scholar), CPD could begin conversion of the pro-TRH intermediates in vivo; however, these effects would be transient as the intermediates passed to the mature granule and a build-up of intermediates could occur.Cold exposure stimulates the hypothalamic-pituitary-thyroid axis strongly (26Arancibia S. Rage F. Astier H. Tapia-Arancibia L. Neuroendocrinology. 1996; 64: 257-267Google Scholar). In rats, cold temperatures increase TRH mRNA levels in the hypothalamic paraventricular nucleus (46Zoeller R.T. Kabeer N. Albers H.E. Endocrinology. 1990; 127: 2955-2962Google Scholar), augment TRH release from the median eminence (47Arancibia S. Tapia-Arancibia L. Assenmacher I. Astier H. Neuroendocrinology. 1983; 37: 225-228Google Scholar), stimulate TSH secretion from pituitary, and potentiate T4 and T3 contents in blood (48Rondeel J.M.M. de Greef W.J. Hop C.J. Rowland D.L. Visser T.J. Neuroendocrinology. 1991; 54: 477-481Google Scholar). In our studies, basal levels of serum TSH and T4 are similar among WT, heterozygous, and Cpefat/fatmice. A similar relationship has been reported for serum T4concentrations in homozygotes and lean controls (7Leiter E.H. Kintner J. Flurkey K. Beamer W.G. Naggert J.K. Endocrine. 1999; 10: 57-66Google Scholar). AlthoughCpefat/fat animals respond to cold, they cannot maintain their core body temperatures or sustain their endocrine responses. The already depressed hypothalamic TRH levels in theCpefat/fat mice are reduced further by an additional 38% with cold exposure. Although TRH neurons reside in several different hypothalamic areas, only the paraventricular nucleus controls anterior pituitary function (50Lechan R.M. Jackson I.M.D. Endocrinology. 1982; 111: 55-65Google Scholar). The large reduction of TRH stores in response to cold suggests that much of the fully processed TRH is located in a readily releasable pool of peptide and that substantial quantities of TRH are released from the hypophyseal neurons. Together, these findings demonstrate that the hypothalamic-pituitary-thyroid axis is impaired in its response to cold and that the TRH processing deficit in theCpefat/fat mouse may contribute to this deficiency.The fat/fat mouse was identified initially at Jackson Laboratories as being obese, diabetic, and infertile (5Coleman D.L. Eichler E.M. J. Hered. 1990; 81: 424-427Google Scholar). Although the regulation of each of these physiological processes is complex, the deficiency in pro-TRH conversion to TRH may contribute to its obesity and diabetes. For instance, disruption of the TRH gene in mice produces alterations in insulin secretion and hyperglycemia (51Yamada M. Saga Y. Shibusawa N. Hirato J. Murakami M. Iwasaki T. Hashimoto K. Satoh T. Wakabayashi K. Taketo M.M. Mori M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10862-10867Google Scholar). Thyroid hormone influences oxygen consumption and metabolism in mammals, and changes in hypothalamic-pituitary-thyroid function can affect weight gain and appetite (52Ahima R.S. Prabakaran D. Mantzoros C. Qu D. Lowell B. Maratos-Flier E. Flier J.S. Nature. 1996; 382: 250-252Google Scholar, 53Nillni E.A. Vaslet C. Harris M. Hollenberg A. Bjorbak C. Flier J.S. J. Biol. Chem. 2000; 275: 36124-36133Google Scholar). In addition, the TSH response to TRH stimulation is increased in obesity (49Donders S.H. Pieters G.F. Heevel J.G. Ross H.A. Smals A.G. Kloppenborg P.W. J. Clin. Endocrinol. Metab. 1985; 61: 56-59Google Scholar). In our studies the TSH response to cold exposure was more robust in the mutants than in the WT animals. This enhanced responsivity in the presence of reduced hypothalamic tissue stores of TRH suggests that TRH levels in hypophyseal blood may be low in the Cpefat/fatmice and that the TRH receptor may be up-regulated on the pituitary. Similar to cold exposure, these circumstances could render this mutant unable to respond normally to metabolic and other challenges. Inasmuch as TRH is found in pancreas, gastrointestinal tract, pineal gland, neurohypophysis, and many regions of the central nervous system (10Reichlin S. Wilson J.D. Foster D.W. Kronenberg H.M. Larsen P.R. Williams Textbook of Endocrinology. 9th Ed. W. B. Saunders Co., Philadelphia, PA1998: 165-248Google Scholar), a deficiency in pro-TRH processing could affect the physiology in a number of additional organs and systems. Neuropeptides and peptide hormones are first biosynthesized as precursors that must undergo a series of conversions to become biologically active (1Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Google Scholar, 2Seidah N.G. Chrétien M. Brain Res. 1999; 848: 45-62Google Scholar). Typically, maturation of these precursors begins with limited proteolysis. Excision of the pro-peptide usually occurs at monobasic, dibasic, or tetrabasic residues where subtilisin-like processing enzymes cleave the precursor on the C-terminal side of these amino acids. The endoproteolysis is usually followed by the sequential removal of the basic amino acid(s) residues by a carboxypeptidase-like enzyme (3Fricker L.D. Ann. Rev. Physiol. 1988; 50: 309-321Google Scholar). In some situations, additional modifications can occur in the form of N-terminal acetylation or pyroglutamate formation, sulfation, and C-terminal amidation (4Eipper B.A. Stoffers D.A. Mains R.E. Ann. Rev. Neurosci. 1992; 15: 57-85Google Scholar). These alterations usually serve to yield a peptide that is both biologically active and resistant to degradation. Further evidence that peptide processing is physiologically relevant has come from a mouse identified at the Jackson Laboratories. This animal was reported to be obese, diabetic, and infertile because of a spontaneous mutation in the fat gene (5Coleman D.L. Eichler E.M. J. Hered. 1990; 81: 424-427Google Scholar). Subsequent studies have shown that the fat/fat mouse has a single point mutation in the carboxypeptidase E (CPE) 1The abbreviations used are: CPE, carboxypeptidase E; CPD, carboxypeptidase D; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; IR, immunoreactivity; PC, prohormone convertase; RIA, radioimmunoassay; T3, triiodothyronine; T4, thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. 1The abbreviations used are: CPE, carboxypeptidase E; CPD, carboxypeptidase D; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; IR, immunoreactivity; PC, prohormone convertase; RIA, radioimmunoassay; T3, triiodothyronine; T4, thyroxine; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. gene, and hence, this mutation has been termed Cpefat (6Naggert J.K. Fricker L.D. Varlamov O. Nishina P.M. Rouille Y. Steiner D.F. Carroll R.J. Paigen B.J. Leiter E.H. Nat. Genet. 1995; 10: 135-142Google Scholar). A Ser202 to Pro202 transition renders the enzyme catalytically inactive and subject to rapid degradation soon after synthesis. With respect to the diabetes in theCpefat/fat mouse, the mechanism underlying this dysfunction has been partially attributed to a deficiency in converting pro-insulin intermediates to insulin. To date, the mechanisms underlying the obesity in theCpefat/fat mouse are not well understood. The reason for these circumstances is probably because of the multifactorial nature of the regulation of food intake, absorption, and caloric utilization (7Leiter E.H. Kintner J. Flurkey K. Beamer W.G. Naggert J.K. Endocrine. 1999; 10: 57-66Google Scholar, 8Fricker L.D. Leiter E.H. Trends Biochem. Sci. 1999; 24: 390-393Google Scholar). In the latter case, the control of metabolism may be especially important. One hormonal system that plays an important role in the regulation of metabolism is the hypothalamic-pituitary-thyroid axis that is controlled by thyrotropin-releasing hormone (TRH). Hypophysiotrophic pro-TRH is synthesized in the hypothalamic paraventricular nucleus, and it must undergo a number of different processing steps to yield mature TRH (9Nillni E.A. Sevarino K.A. Endocrinol. Rev. 1999; 20: 599-648Google Scholar). Upon stimulation, TRH is released from median eminence nerve terminals into the hypophyseal blood where it is transported to the pituitary to stimulate the biosynthesis and secretion of thyroid-stimulating hormone (TSH; see Ref. 10Reichlin S. Wilson J.D. Foster D.W. Kronenberg H.M. Larsen P.R. Williams Textbook of Endocrinology. 9th Ed. W. B. Saunders Co., Philadelphia, PA1998: 165-248Google Scholar). TSH, in turn, is carried in blood to the thyroid where it stimulates thyroid hormone biosynthesis and release. Besides this role, TRH can also exert some control over the release of prolactin, growth hormone, vasopressin, and insulin, as well as the classic neurotransmitters, norepinephrine and epinephrine. Furthermore, TRH is present in brain regions outside of the hypothalamus where it may serve as a neurotransmitter or neuromodulator. In previous reports, it has been shown that the pro-TRH is cleaved endoproteolytically by two different members of the prohormone convertase (PC) family of enzymes, PC1/3 and PC2 (11Schaner P. Todd R.B. Seidah N.G. Nillni E.A. J. Biol. Chem. 1997; 272: 19958-19968Google Scholar). In this scheme, PC1/3 appears to be primarily responsible for most of the major cleavage events. By contrast, although PC2 can perform many of these same conversions, this enzyme is required for the formation of the pEH24 peptide, and it is specifically involved in the processing of prepro-TRH178–199 to generate the pFQ7 and pSE14 peptides (11Schaner P. Todd R.B. Seidah N.G. Nillni E.A. J. Biol. Chem. 1997; 272: 19958-19968Google Scholar, 12Nillni E.A. Aird F. Seidah N.G. Todd R.B. Koenig J.I. Endocrinology. 2001; 142: 896-906Google Scholar). Because PC1/3 and PC2 seem to cleave the pro-TRH on the C-terminal side of dibasic residues, these basic amino acids must be removed from the pro-TRH intermediates for the TRH to be active biologically. An enzyme that has been hypothesized to produce these conversions is CPE. Because theCpefat/fat mouse has a mutation in this enzyme, this animal should allow us to examine this hypothesis and determine whether this mutation exerts any effects on hypothalamic-pituitary-thyroid function. DISCUSSIONCPE is an exopeptidase that is responsible for removing basic amino acids from the C-terminal of proteins and peptides (3Fricker L.D. Ann. Rev. Physiol. 1988; 50: 309-321Google Scholar). A point mutation in the CPE gene (e.g. Ser202to Pro202 transition) is sufficient to reduce the efficiency" @default.
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• W2058421805 title "Deficiencies in Pro-thyrotropin-releasing Hormone Processing and Abnormalities in Thermoregulation in CpeMice" @default.
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