**1. Introduction**

As primary drivers of growth and proliferation at the somatic, tissue and cellular levels, the insulin-like growth factors (IGFs) must have tightly regulated activity—in terms of amount, timing, and spatial specificity and coordination. The "somatomedin hypothsies", the original framework of understanding IGF-I production and action, has undergone considerable development with time, as additional layers of complexity and nuance have been elucidated [1]. This review summarizes traditionally recognized regulators of IGF production and action in health and disease, and adds ye<sup>t</sup> another: glucose regulated protein 94 (GPR94). GRP94, a ubiquitously expressed chaperone in the endoplasmic reticulum, is required for the proper folding and secretion of both IGFs. Although insulin, the other member of the IGF hormone family, shares similarities with the IGFs structurally (including approximately 50% amino acid homology to the IGFs), in their receptors and signaling, and is itself chaperoned by GRP94, this review will focus on the IGFs. By providing a novel nexus of regulating IGF production, GRP94 and its alterations serve as a potentially new mechanism of dysregulated growth, such as idiopathic short stature and cancer, and thereby may lead to new therapeutic interventions.

#### **2. Regulators of IGF Levels Clinically**

Insulin-like growth factor (IGF)-I is made throughout the body, though ~70% of circulating levels are of hepatic origin. Clinically, measurement of circulating IGF-I concentration is used most commonly as a marker of growth hormone (GH) bioactivity. Due to the GH dependence of transcription of the genes encoding IGF-I and its principal circulating partner, IGF binding protein (IGFBP)-3, normal levels of IGF-I and IGFBP-3 generally reflect normal GH activity (i.e., exclude GH deficiency) [2,3]. Further, because serum levels of these molecules do not fluctuate diurnally as does the pulsatile secretion of GH, they serve as convenient indicators of GH secretion that are measurable on a random blood sample. Indeed, circulating concentration of IGF-I was shown in 114 healthy children and adolescents to both correlate with height and reflect spontaneous GH secretion [4]. IGF-I levels are monitored during treatment with exogenous GH to assess adherence and inform dose adjustments [2,3,5]. Conversely, IGF-I levels are also employed in diagnosing and evaluating treatment efficacy for acromegaly, the state of excessive GH secretion [6,7].

Altered nutritional status represents the major non-GH, clinically relevant regulator of IGF-I levels [8]. Inadequate nutrition causes hepatic GH insensitivity, with a primary reduction in IGF-I production despite normal or even elevated levels of GH from loss of the normal IGF-I negative feedback on the pituitary gland and hypothalamus. This has been characterized in patients with anorexia nervosa [9,10], but also can be seen due to malnutrition from food insecurity (e.g., marasmus and kwashiorkor), milder dietary intake inadequacy that fails to meet daily demands, or specific micronutrient deficit like zinc deficiency [8]. Gastrointestinal diseases like Crohn's disease, celiac disease, cystic fibrosis, and gastroesophageal reflux disease also can lower circulating IGF-I levels even in the absence of gastrointestinal symptoms [8]. In the other extreme, obesity blunts GH secretion, ye<sup>t</sup> circulating IGF-I concentrations in obese individuals often are maintained or even higher than in non-obese controls. Although obesity did not associate with higher total IGF-I levels in multiple studies (indeed, an inverse U-shaped association between IGF-I z-score and body mass index (BMI) was shown in a population-based study of more than 6000 adults [11]), IGF-I bioavailability is increased [8].

Circulating IGF-I concentrations are dependent on age, gender, and gonadal status (pubertal status in adolescence, reproductive function in adults, and even whether estrogen replacement in women is administered orally versus transdermally) [12]. Normal IGF-I concentrations rise from infancy through childhood, peak during puberty (the pubertal growth spurt), and gradually decline throughout adulthood [13,14]. Alterations in circulating IGF-I concentration can also result from hepatic disease, renal dysfunction, and diabetes mellitus. Even controlling for these factors, various IGF-I assays often produce discordant results [15], leading to calls for harmonization of IGF-I assays to prevent diagnostic misclassifications and to allow meaningful inter-study comparisons of results in the literature [2,3,16].

Although closely related to IGF-I, IGF-II shows a different pattern. In healthy newborns, IGF-II concentrations are highest in the fetus, about half that of adult levels, which are reached by 1 year of age and persist through life [17]. In contrast, rodent IGF-II expression declines early postnatally, such that rodent models cannot serve to elucidate the physiologic function of IGF-II persistence in humans. Nonetheless, it is clear IGF-II plays an important role, especially in prenatal growth. In humans, the *IGF2* gene is imprinted and paternally expressed. DNA hypomethylation in the region of the *IGF2* gene that reduces paternal *IGF2* expression presents clinically with Silver–Russell syndrome, which is characterized by both prenatal and postnatal growth failure, often with body segmen<sup>t</sup> asymmetry [18]. *IGF2* overexpression (biallelic expression from relaxation or loss of imprinting) can result in Beckwith–Wiedemann syndrome, an overgrowth syndrome that also affects both pre- and postnatal growth, can include disproportionate growth (such as macroglossia and hemihypertrophy), and is associated with increased risk of embryonal tumors [19].

#### **3. Molecular Regulation of IGF-I Production**

IGF action can be modulated at the level of hormone (or autocrine/paracrine) production, hormone bioavailability, or receptor density and activity. Transcription of the *Igf1* gene, on chromosome *12q23.2*, is regulated by GH, in a mechanistic axis termed the "somatomedin hypothesis", which has undergone considerable refinement over the years [20,21]. The GH receptor is a paradigmal cytokine receptor, whose activation recruits the tyrosine kinase Janus kinase 2 (JAK2). This, in turn, activates the signal transducers and activators of transcription, especially STAT5b [22], a transcription factor that stimulates transcription of the IGF genes. Apart from JAK2, the GH receptor also directly activates the Src tyrosine kinase pathway, the MAP kinase pathway, the PI3K/Akt pathway, and the mTOR pathway [23–25]. Naturally occurring and experimentally induced mutations have shed light on the specificity of second messenger recruitment and the specificity of outcomes conferred by them. Mutations in the GH receptor or in STAT5b are known to impair IGF production and lead to patients with primary IGF-deficient growth failure [26,27].

The *Igf1* gene encodes the 7.6 kD, single chain 70 amino acid polypeptide, that is cross-linked by disulfide bridges [28]. The *Igf2* gene, on chromosome 11p15.5, encodes the single chain 67 amino acid polypeptide [29] and is primarily regulated by imprinting. As discussed below, IGF-I and IGF-II production is determined not only by their transcriptional regulation, but also by interactions with dedicated molecular chaperones.

IGF bioavailability is primarily regulated via a family of six high-a ffinity IGF binding proteins (IGFBPs). Additional lower-a ffinity IGF binding proteins (named IGFBP-related proteins (IGFBPrPs)) were found by in silico searches for homology to the known IGFBPs; many of these molecules were previously known in other contexts, serving roles in normal or neoplastic growth [30]. The IGFBPs prolong the circulating half-life of IGF, transport the IGFs to target cells, and modulate the interaction of the IGFs with their surface membrane receptors via competitive inhibition. Local proteases, such as metalloproteinase pregnancy associated plasma protein A2 (PAPPA2), cleave the IGFBP, releasing the IGF for binding and activation of its receptor [31]. Of note, the IGFBPs have been found to perform various IGF-independent functions as well [30].

The actions of both IGF-I and IGF-II is mediated via the type 1 IGF receptor (IGF1R), an α2β2 transmembrane tyrosine kinase receptor that upon ligand binding, autophosphorylates and phosphorylates signaling pathways such as MAPK and PI3K/Akt [32]. IGF1R bears a high degree of homology to the insulin receptor, and αβ-hemireceptors of the two can form functioning hybrid receptors [33]. IGF1R signaling is regulated by internalization of bound receptors into clathrin-coated pits [34]. Phosphatases like SHP2 also can limit IGF1R signaling [35]. In contrast to IGF1R, the type 2 IGF receptor binds only IGF-II with high a ffinity, does not possess any recognizable signal transduction mechanism, and is identical to the cation-independent mannose-6-phosphate (CIM6P) receptor, a protein involved in intracellular lysosomal targeting [36]. Given the complexities of the system, an IGF-IR kinase receptor activation assay has been developed to measure IGF-1R stimulating activity (phosphorylation of tyrosine residues of the IGFIR) as a means of assessing the net e ffects of the system's multiple players in various conditions [37].

Whereas the transcriptional and translational regulation of IGF production follow usual paradigms, the post-translational regulation of IGFs has unique features. First, as discussed below, maturation of IGF-I depends on the activity of GRP94, and without it IGF-I does not complete its biosynthesis and is not secreted [38]. This chaperone interaction provides a new element of regulation outside the "standard" GH system. Second, as mentioned above, the complexes of IGF-I with the binding proteins are important for IGF-I function.
