Mandy Sawitzky

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I).

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

Lack of Dietary Carbohydrates Induces Hepatic Growth Hormone (GH) Resistance in Rats

GH is a well established regulator of growth, lipid, and glucose metabolism and therefore important for fuel utilization. However, little is known about the effects of macronutrients on the GH/IGF system. We used low-carbohydrate/high-fat diets (LC-HFD) as a model to study the impact of fat, protein, and carbohydrates on the GH/IGF-axis; 12-wk-old Wistar rats were fed either regular chow, a moderate, protein-matched LC-HFD, or a ketogenic LC-HFD (percentage of fat/protein/carbohydrates: chow, 16.7/19/64.3; LC-HF-1, 78.7/19.1/2.2; LC-HF-2, 92.8/5.5/1.7). After 4 wk, body and tibia length, lean body mass, and fat pad weights were measured. Furthermore, we investigated the effects of LC-HFD on 1) secretion of GH and GH-dependent factors, 2) expression and signaling of components of the GH/IGF system in liver and muscle, and 3) hypothalamic and pituitary regulation of GH release. Serum concentrations of IGF-I, IGF binding protein-1, and IGF binding protein-3 were lower with LC-HF-1 and LC-HF-2 (P < 0.01). Both LC-HFD-reduced hepatic GH receptor mRNA and protein expression, decreased basal levels of total and phosphorylated Janus kinase/signal transducers and activators of transcription signaling proteins and reduced hepatic IGF-I gene expression. Hypothalamic somatostatin expression was reduced only with LC-HF-1, leading to increased pituitary GH secretion, higher IGF-I gene expression, and activation of IGF-dependent signaling pathways in skeletal muscle. In contrast, despite severely reduced IGF-I concentrations, GH secretion did not increase with LC-HF-2 diet. In conclusion, lack of carbohydrates in LC-HFD induces hepatic GH resistance. Furthermore, central feedback mechanisms of the GH/IGF system are impaired with extreme, ketogenic LC-HFD.

Increased fat mass, decreased myofiber size, and a shift to glycolytic muscle metabolism in adolescent male transgenic mice overexpressing IGFBP-2

To elucidate the functional role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) for in vivo skeletal muscle growth and function, skeletal muscle cellularity and metabolism, expression of signal molecules, and body growth and composition were studied in a transgenic mouse model overexpressing IGFBP-2. Postnatal growth rate of transgenic mice was reduced from day 21 of age by 6-8% compared with nontransgenic controls. At 10 wk of age body lean protein and moisture percentages were lower, whereas fat percentage was higher in IGFBP-2 transgenic mice. Muscle weights were reduced (-13% on day 30 of age, -14% on day 72), which resulted from slower growth of myofibers in size but not from decreases in myofiber number. The reduction in muscle mass was associated with lower total DNA, RNA, and protein contents as well as greater DNA/RNA and protein/RNA ratios. The percentage of proliferating (Ki-67-positive) nuclei within myofibers was reduced (3.4 vs. 5.8%) in 30-day-old transgenic mice. These changes were accompanied by slight reductions in specific p44/42 MAPK activity (-18% on day 72) and, surprisingly, by increased levels of phosphorylated Akt (Ser(473)) (+25% on day 30, +66% on day 72). The proportion of white glycolytic fibers (55.9 vs. 53.5%) and the activity of lactate dehydrogenase (+8%) were elevated in 72-day-old transgenic mice. Most of the differences observed between transgenic and nontransgenic mice were more pronounced in males. The results suggest that IGFBP-2 significantly inhibits postnatal skeletal myofiber growth by decreasing myogenic proliferation and protein accretion and enhances glycolytic muscle metabolism.

Serum IGF-I Is Not a Reliable Pharmacodynamic Marker of Exogenous Growth Hormone Activity in Mice

Serum IGF-I is a well-established pharmacodynamic marker of GH administration in humans and has been used for this purpose in animal studies. However, its general suitability in wild-type laboratory mice has not been demonstrated. Here we show that treatment with recombinant human GH (rhGH) in four different strains of laboratory mice increases body weight, lean body mass, and liver weight but does not increase hepatic expression and release of IGF-I. In contrast and as expected, hypophysectomized rats show a rapid increase in serum IGF-I after rhGH administration. The lack of IGF-I up-regulation in mice occurs despite hepatic activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and is not explained by GH dose, route of administration, origin of GH (i.e. recombinant human, bovine, and murine GH), treatment duration, genetic background, sex, or formation of neutralizing antibodies. Effects on other components of the GH/IGF pathway were highly influenced by genetic background and sex but not consistently affected by rhGH treatment. We conclude that IGF-I is not a reliable indicator of the biological effects of exogenous GH treatment in genetically and pharmacologically unmodified mice. We speculate that IGF-I release is already maximal in these animals and cannot be further increased by exogenous GH treatment. This is also suggested by the observation of restored IGF-I up-regulation in isolated murine hepatocytes after rhGH treatment. Total body weight, lean body mass, and liver weight may be more reliable phenotypic indicators in these models.

Chronic Growth Hormone Excess Is Associated with Increased Aldosterone: A Study in Patients with Acromegaly and in Growth Hormone Transgenic Mice:

Acromegaly is a disease characterized by chronic growth hormone (GH) excess. Since hypertension is a common finding in patients with acromegaly, interactions between GH and the renin-angiotensin-aldosterone system (RAAS) are under controversial debate. We examined GH, IGF-I, aldosterone, and renin in a well-defined group of acromegalic patients before and after cure by surgery. In addition, we analyzed the impact of chronic GH excess on the RAAS in mouse models over-expressing GH alone (G) or in combination with insulin-like growth factor–binding protein-2 (IGFBP-2; GB). Normalization of GH secretion after cure by surgery was accompanied by significant decreases of serum aldosterone in acromegalic patients (pre-op: 96.5 ± 37.1 pg/mL, post-op: 41.3 ± 28.2 pg/ mL; P < 0.001; n = 13), but renin concentrations were unaffected. In addition, aldosterone concentrations were positively correlated to GH levels (Spearman r = 0.39; P = 0.025; n = 26). To further study this association, we analysed two transgenic mouse models and found a similar relationship between GH and aldosterone in G mice, which showed about 3-fold elevated serum aldosterone levels in comparison to non-transgenic controls (males: 442 ± 331 pg/mL vs. 151 ± 84 pg/mL; P = 0.002; n ≥ 12; females: 488 ± 161 pg/mL vs. 108 ± 125 pg/mL; P = 0.05; n ≥ 4). Expression of aldosterone synthase was similar in adrenal glands of C and G mice. Aldosterone levels in G and GB mice of both genders were not different, indicating that the elevated aldosterone was due to GH excess and not caused by elevated IGF-I, which is known to be blocked by IGFBP-2 overexpression. Also in the mouse models, changes in aldosterone were independent from renin. In summary, we show that chronic GH excess is associated with increased aldosterone in humans and mice. GH-induced increases of aldosterone potentially contribute to the increased cardiovascular risk in acromegalic patients. The underlying mechanism is likely to be independent of renin, excess IGF-I, or adrenal aldosterone synthase expression.

Decreased p44/42 Mitogen-Activated Protein Kinase Phosphorylation in Gender- or Hormone-Related But Not during Age-Related Adrenal Gland Growth in Mice

Postnatal growth of the mouse adrenal gland shows a characteristic gender-dependent pattern, resulting in an almost 2-fold higher adrenal weight in 11-wk-old female vs. male mice. We demonstrated that the higher weight of the adrenal glands in female mice is due to a significantly (P < 0.05) increased growth rate in female mice and a shorter growth phase of the adrenal glands in male mice (P < 0.05). To address the signaling mechanisms underlying these differential growth patterns, we evaluated the phosphorylation levels of p44/42 and p38 MAPK. In female mice, age-dependent reductions of p38 MAPK phosphorylation were found between wk 3 and 9 (47% reduction; P < 0.05). At the age of 11 wk, the p38 MAPK phosphorylation level in female adrenal glands was about 60% lower than in the male counterparts (P < 0.01). Similarly, the phosphorylation level of p44/42 MAPK was 50% lower in female adrenal glands (P < 0.001). Reduced activation of p44/42 MAPK was also observed after growth stimulation of the adrenal glands in male mice after ACTH treatment (-36%; P < 0.001) or by expression of a GH transgene (-34%; P < 0.001), whereas p38 MAPK, JNK, or PDK1 activation was unaffected. From our findings in three independent mouse models where partial deactivation of p44/42 MAPK was observed under conditions of elevated growth, we suggest a function of p44/42 MAPK for adrenal growth and a role of p44/42 MAPK for the integration of different endocrine stimuli.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

P-64 Decreased p44/42 MAPK phosphorylation in gender- or hormone-related but not during age-related adrenal gland growth in mice

Postnatal growth of the mouse adrenal gland shows a characteristic gender-dependent pattern, resulting in an almost two-fold higher adrenal weight in 11-week-old female versus male mice. We demonstrated that the higher weight of the adrenal glands in female mice is due to a significantly (p<0.05) increased growth rate in female mice and a shorter growth phase of the adrenal glands in male mice (p<0.05). To address the signaling mechanisms underlying these differential growth patterns, we evaluated the phosphorylation levels of p44/42 and p38 MAPK. In female mice, age dependent reductions of p38 MAPK phosphorylation were found between week 3 and week 9 (47% reduction; p<0.05). At the age of 11 weeks the p38 MAPK phosphorylation level in female adrenal glands was about 60% lower than in the male counterparts (p<0.01). Similarly the phosphorylation level of p44/42 MAPK was 50% lower in female adrenal glands (p<0.001). Reduced activation of p44/42 MAPK was also observed after growth stimulation of the adrenal glands in male mice after ACTH treatment (-36%; p<0.001) or by expression of a growth hormone transgene (-34%; p<0.001), while p38 MAPK, JNK or PDK1 activation were unaffected. From our findings in three independent mouse models where partial deactivation of p44/42 MAPK was observed under conditions of elevated growth we suggest a function of p44/42 MAPK for adrenal growth and a role of p44/42 MAPK for the integration of different endocrine stimuli.

OR12,67 Age-dependent control of Akt phosphorylation in muscles from IGFBP-2 transgenic mice as a potential target for the metabolic effects of IGFBP-2

proliferation assays, the dose-response effect induced by the human growth hormone mutants did not differ from the effects induced by wildtype hGH. Besides, wildtype hGH and the four hGH variants were able to activate STAT5 to the same extent. However, it was observed that production and secretion of the human growth hormone variants were significantly lower than wildtype hGH, as determined by fluorescent sandwich-type immunoassays and confirmed with Western blot analysis. Particularly low secretion was assessed for P89L and V110F, which showed only 15% and 24% of wildtype hGH concentration, respectively. Co-expression of wildtype and mutant human growth hormone furthermore suggested inhibition of wildtype hGH production by mutant hGH. These results indicate that the short stature in patients with one of the four heterozygous missense mutations might be caused by an intracellular event within the somatotropic cells in the anterior pituitary leading to reduced production and/or secretion of the hGH variants as well as of wildtype hGH.