**2. Effects of L-PHP on pancreatic insulin secretion**

Beyond regulating TSH, L-PHP is also found to be involved in the regulation of neuronal growth [9], facilitating spinal cord injury recovery [10], appetite control [11], and alcohol consumption [12]. The most important role of L-PHP is considered to be its regulation of blood glucose levels *in vivo*, presumably *via* the CNS [6, 13-16]. L-PHP's anti-hyperglycemia function was identified by eliminating pituitary-thyroid axis by a hypophysectomy, which also eliminated other hormones released from pituitary, and suggests its anti-hyperglycemia function beyond its activation in CNS [17]. In another experiment, pancreatic beta cells were destroyed by Streptozotocin and CNS administration of L-PHP failed to reverse high blood glucose, supporting this notion of function outside of CNS activation. L-PHP regulating blood glucose may have a direct effect in pancreatic beta cell instead of *via* CNS or thyroid hormone, which was supported by application of thyroid hormone in hypothyroidism of hyperglycemic animal but did not reverse high blood glucose. Blood glucagon and insulin level was increased by intravenous injection of L-PHP in rabbit [18] and cultured fetal islet identified L-PHP expression by a quantitative analysis [19] which supports the possibility of L-PHP's direct effect on pancreatic beta cell function.

Pancreatic L-PHP can stimulate pancreatic endocrine function and/or endocrine cell develop‐ ment. The mechanisms as to how L-PHP regulates pancreatic β-cell development have not been identified. Gathering evidence from *in vivo* and *in vitro*, we propose that L-PHP may modulate insulin secretion directly when glucose stimulates β-cell, which was demonstrated in isolated perfusion of fresh islets [20] and islet cell lines (Fig. 1). The mechanisms may relate to L-PHP regulating glucagon-containing (alpha) cell secretion resulting in eliminating somatostatin (r-cells) and inhibiting insulin production. A clinical study in hyperparathyroid‐ ism patients showed that L-PHP application to these patients significantly elevated serum levels of insulin and glucagon and it also had a dose-dependent inhibition of carbacholstimulated amylase secretion, suggesting a role for L-PHP in the paracrine regulation of exocrine as well as endocrine pancreatic secretion.

observation of ten-fold lower L-PHP in pups of diabetic rat followed by a postnatal day 5 elevation of L-PHP reducing blood glucose levels [22] suggest that L-PHP expression during β-cell development is important and it may prevent diabetes from developing in later life.

(b) Fig. (1). Insulin levels in βTC-6 (a mouse derived pancreatic β cell line) cell extracts and medium after exposure to L-PHP (TRH) Cells were cultured for 24 hours with or without L-PHP(n=6 each group). Culture Medium was collected and harvested cells were extracted by 5% TCA. Insulin content and secretion were measured by ELISA. Insulin content was normalized relative to protein concentration (mg/ml) in the cell extracts. L-PHP treated cells contained greater levels of

**Figure 1.** Insulin levels in βTC-6 (a mouse derived pancreatic β cell line) cell extracts and medium after exposure to TRH Cells were cultured for 24 hours with or without TRH (n=6 each group). Culture Medium was collected and har‐ vested cells were extracted by 5% TCA. Insulin content and secretion were measured by ELISA. Insulin content was normalized relative to protein concentration (mg/ml) in the cell extracts. L-PHP treated cells contained greater levels

insulin in cell extracts A and culture medium B vs controls. (From reference #25, with permission).

of insulin in cell extracts A and culture medium B vs controls. (From reference #25, with permission)

(a)

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The L-PHP receptor consists of two major sub-types (R1 and R2, recently identified third type). Using RT-PCR, receptor R1 is identified as expressed in HIT-T15 (HIT) cells, a hamster clonal ß-cell line [23], and mouse pancreatic islets, but expression of R2 is not found. R2 was identi‐ fied as expressed predominantly in the CNS, but not other tissue. By northern blot analysis it was found that R1 in pancreas is of 3.7-kb size and shares 93.3% homology with that in the pituitary. Evaluation of R1 function by receptor affinity found various kDa values in ß –cells [23]. ß –cell intracellular calcium concentration was significantly increased by L-PHP and removal of extracellular calcium does not change this effect [24]. Our group work has shown

L-PHP protects pancreatic tissue from damage and toxins like the reduction of glycodeoxy‐ cholic acid. Evidence suggests that L-PHP plays a critical role in β-cell maturation. During the phase of pancreatic development, which includes high levels of L-PHP expression in early pancreatic β-cell development, dexamethasone treatment eliminated the L-PHP peak and resulted in retarded β-cell development [21]. Also, newborn rats were found to have reduced L-PHP levels due to maternal diabetes caused by streptozotocin (STZ) injection [22]. The granules resemble β cells. However, at E16, L-PHP expression was found and thereafter, high expression of molecules such as Glut2 and Pdx-1, which are necessary for insulin production, maturation and full insulin cell function, were found in the insulin and L-PHP positive cells. L-PHP's significant expression coincides with factors for insulin production, maturation and insulin cell development suggesting that L-PHP is critical for insulin cells as they become

Beyond regulating TSH, L-PHP is also found to be involved in the regulation of neuronal growth [9], facilitating spinal cord injury recovery [10], appetite control [11], and alcohol consumption [12]. The most important role of L-PHP is considered to be its regulation of blood glucose levels *in vivo*, presumably *via* the CNS [6, 13-16]. L-PHP's anti-hyperglycemia function was identified by eliminating pituitary-thyroid axis by a hypophysectomy, which also eliminated other hormones released from pituitary, and suggests its anti-hyperglycemia function beyond its activation in CNS [17]. In another experiment, pancreatic beta cells were destroyed by Streptozotocin and CNS administration of L-PHP failed to reverse high blood glucose, supporting this notion of function outside of CNS activation. L-PHP regulating blood glucose may have a direct effect in pancreatic beta cell instead of *via* CNS or thyroid hormone, which was supported by application of thyroid hormone in hypothyroidism of hyperglycemic animal but did not reverse high blood glucose. Blood glucagon and insulin level was increased by intravenous injection of L-PHP in rabbit [18] and cultured fetal islet identified L-PHP expression by a quantitative analysis [19] which supports the possibility of L-PHP's direct

Pancreatic L-PHP can stimulate pancreatic endocrine function and/or endocrine cell develop‐ ment. The mechanisms as to how L-PHP regulates pancreatic β-cell development have not been identified. Gathering evidence from *in vivo* and *in vitro*, we propose that L-PHP may modulate insulin secretion directly when glucose stimulates β-cell, which was demonstrated in isolated perfusion of fresh islets [20] and islet cell lines (Fig. 1). The mechanisms may relate to L-PHP regulating glucagon-containing (alpha) cell secretion resulting in eliminating somatostatin (r-cells) and inhibiting insulin production. A clinical study in hyperparathyroid‐ ism patients showed that L-PHP application to these patients significantly elevated serum levels of insulin and glucagon and it also had a dose-dependent inhibition of carbacholstimulated amylase secretion, suggesting a role for L-PHP in the paracrine regulation of

L-PHP protects pancreatic tissue from damage and toxins like the reduction of glycodeoxy‐ cholic acid. Evidence suggests that L-PHP plays a critical role in β-cell maturation. During the phase of pancreatic development, which includes high levels of L-PHP expression in early pancreatic β-cell development, dexamethasone treatment eliminated the L-PHP peak and resulted in retarded β-cell development [21]. Also, newborn rats were found to have reduced L-PHP levels due to maternal diabetes caused by streptozotocin (STZ) injection [22]. The

functionally mature during early development.

156 Glucose Homeostasis

effect on pancreatic beta cell function.

exocrine as well as endocrine pancreatic secretion.

**2. Effects of L-PHP on pancreatic insulin secretion**

Fig. (1). Insulin levels in βTC-6 (a mouse derived pancreatic β cell line) cell extracts and medium after exposure to L-PHP (TRH) Cells were cultured for 24 hours with or without L-PHP(n=6 each group). Culture Medium was collected and harvested cells were extracted by 5% TCA. Insulin content and secretion were measured by ELISA. Insulin content was normalized relative to protein concentration (mg/ml) in the cell extracts. L-PHP treated cells contained greater levels of insulin in cell extracts A and culture medium B vs controls. (From reference #25, with permission). **Figure 1.** Insulin levels in βTC-6 (a mouse derived pancreatic β cell line) cell extracts and medium after exposure to TRH Cells were cultured for 24 hours with or without TRH (n=6 each group). Culture Medium was collected and har‐ vested cells were extracted by 5% TCA. Insulin content and secretion were measured by ELISA. Insulin content was normalized relative to protein concentration (mg/ml) in the cell extracts. L-PHP treated cells contained greater levels of insulin in cell extracts A and culture medium B vs controls. (From reference #25, with permission)

(b)

observation of ten-fold lower L-PHP in pups of diabetic rat followed by a postnatal day 5 elevation of L-PHP reducing blood glucose levels [22] suggest that L-PHP expression during β-cell development is important and it may prevent diabetes from developing in later life.

The L-PHP receptor consists of two major sub-types (R1 and R2, recently identified third type). Using RT-PCR, receptor R1 is identified as expressed in HIT-T15 (HIT) cells, a hamster clonal ß-cell line [23], and mouse pancreatic islets, but expression of R2 is not found. R2 was identi‐ fied as expressed predominantly in the CNS, but not other tissue. By northern blot analysis it was found that R1 in pancreas is of 3.7-kb size and shares 93.3% homology with that in the pituitary. Evaluation of R1 function by receptor affinity found various kDa values in ß –cells [23]. ß –cell intracellular calcium concentration was significantly increased by L-PHP and removal of extracellular calcium does not change this effect [24]. Our group work has shown R1 expression in rat-derived β-cell lines as well as whole pancreas that included nonislet tissue [25]. R1 receptor was also found to associate with EGF receptor function called cross linking [25] (Fig. 2).

genes are found to be regulated by L-PHP, 29 genes in the pancreas and 31 genes in rat derived pancreatic β-cell line, INS-1 cells. These genes include Ca2+channel enhancers (Ca2+/calmo‐ dulin-dependent protein kinase, type I and II), G-protein coupling receptor related genes (GPCR kinase 4 and 5, transducin-β 1 subunit,Arrestin-β1, transducin-β1), Protein kinases (serine/threonine kinase-3,PKC β, PCTAIRE-3, v-mos), proliferation or differentiation signal transduction related genes (MAPK3, growth factor receptor-bound protein 2, n-myc, GAP-43) and down-regulated pro-apoptotic Bax gene. Genes relative to insulin secretion are signifi‐ cantly increased by L-PHP including N-methyl-D-aspartate receptor-2A, GABA-A receptor, RAB2, Ras-related GTPase and ADP ribosylation factor 1 and 5. The differential gene expres‐ sion between β-cells and total pancreatic tissue in response to L-PHP shows that of the 36 genes that are initiated and 36 genes that are turned off relative to signal transduction. In rat pancreas 6 genes were initiated and 14 genes were turned off, with one initiating the anti-apoptotic BcLX gene. While in rat INS-1 β cell line only 4 genes were initiated and 4 genes turned off from the 34 signal transduction genes. These significant variations between pure β-cell and entire pancreatic tissue indicate that L-PHP can regulate β-cell function by directly working on βcells or by indirectly altering pancreatic microenvironment to maintain and facilitate β-cell

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response to glucose resulting in a balance *in vivo* of glucose metabolism.

**growth hormone activity in pancreatic islet**

**5. Regulation of β-cell proliferation by signal pathways from L-PHP to**

L-PHP has been reported to stimulate R1 and dissociate the GPCR complex, activating protein kinase C [29] and mitogen-activated protein kinase (MAPK) [29] in both a PKC-dependent and a PKC-independent manner in the neuronal cell lines [30]. These effects may involve activation of tyrosine kinase, which leads to the activation of Ras and MAPK cascade. The signaling pathways initiating from G-coupled L-PHP receptor in activating MAPK may overlap with the receptor tyrosine kinases activating the Ras-MAPK cascade [31, 32]. There is evidence that L-PHP and EGF have overlapping activities [33] leading to the stimulation of tyrosine phosphorylation of EGF receptors in GH3 cells, a pituitary cell line [34]. L-PHP-induced EGF receptor phosphorylation led to the recruitment of adapter protein Grb2 and Shc in GH3 cells. The hypothesis that L-PHP would activate EGF receptors in β cells through multiple pathways is tested, and data indicated that L-PHP trans-activates EGF receptors through several intraand extracellular pathways, which are distinguished from pituitary-derived cell lines. R1 can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic β cells [35]. By initiating R1 G-protein-coupled receptor (GPCR) and dissociated αβγ complex, L-PHP (200nM) activates tyrosine residues at Tyr845, (a known target for Src) and Tyr1068 in the EGF receptor complex in an immortalized mouse β-cell line, βTC-6. Through manipulating the activation of Src, PKC and heparin-binding EGF-like growth factor (HB-EGF) with corresponding individual inhibitors and activators, multiple signal transduction pathways linking L-PHP to EGF receptors in βTC-6 cell lines have been revealed. The pathways include the activation of Src kinase and the release of heparin-binding EGF as a consequence of MMP3 activation. Alternatively, L-PHP inhibited PKC activity by reducing

Fig. (2). In situ hybridization of L-PHP-receptor-1(R1) in rat pancreas A. and D. Dual fluorescent image of rat pancreas. Red indicates insulin immuno-fluorescence; Green indicates R1 in situ hybridization. B. and E show H&E staining for tissue morphology, C and F show dapi for nuclei staining. The large arrows indicate the yellow color, a mixture of green and red represents colocalization of insulin and R1 in islet and the small white arrows indicate the positive staining of R1 in epithelial A. B. and ductal D. E. (From reference #25, with permission). **Figure 2.** In situ hybridization of L-PHP-receptor-1(R1) in rat pancreas A. and D. Dual fluorescent image of rat pan‐ creas. Red indicates insulin immuno-fluorescence; Green indicates R1 in situ hybridization. B. and E show H&E staining for tissue morphology, C and F show dapi for nuclei staining. The large arrows indicate the yellow color, a mixture of green and red represents colocalization of insulin and R1 in islet and the small white arrows indicate the positive stain‐ ing of R1 in epithelial A. B. and ductal D. E. (From reference #25, with permission)
