The Insulin Journey in the Human Body

Abstract

Insulin represents the paramount anabolic hormone and the master regulator of glucose, lipid, and protein metabolism. This chapter describes the sequential stages of the physiologic journey of insulin in the human body, from its synthesis/secretion to its action in peripheral tissues and, ultimately, to its clearance and degradation. These stages include i) insulin synthesis and release from pancreatic beta cells; ii) insulin first-pass metabolism and partial clearance in the liver; iii) insulin action on the vasculature and exit from the capillary beds; iv) insulin action in peripheral and central target tissues (skeletal muscle, adipose tissue, liver, and central nervous system); and v) final insulin degradation in the kidney. Each of these stages is regulated by complex intracellular mechanisms that take place in different tissues and allow for the anabolic actions of insulin. Understanding the abovementioned stages is pivotal to comprehending the clinical consequences of impaired insulin secretion and action, as defects in one or more of these stages can be associated with the development of insulin resistance, metabolic syndrome, and type 2 diabetes mellitus. Additionally, a thorough knowledge of the insulin bodily journey can assist clinicians in therapeutic decision-making for diabetic patients on exogenous insulin therapy in different clinical settings.

1. Introduction

The year 2021 marked the 100th anniversary of the discovery of insulin, which was first isolated from a dog’s pancreas by Frederick Grant Banting and Charles Herbert Best under the directorship of John James Rickard Macleod at the University of Toronto in 1921. Insulin purification was then made possible by James Collip. The Nobel Prize in Physiology or Medicine 1923 was awarded jointly to Frederick Grant Banting and John James Rickard Macleod for the discovery of insulin. This groundbreaking discovery has since saved and improved the lives of millions of people with diabetes worldwide [1].

This chapter aims to describe in detail the sequential stages of the physiologic journey of insulin in the human body, from its synthesis and secretion to its action in central and peripheral target tissues and, ultimately, to its clearance and degradation. Specifically, the chapter will focus on five essential stages of the insulin bodily journey, namely: i) insulin biosynthesis and release from pancreatic beta cells; ii) insulin first-pass metabolism and partial clearance in the liver; iii) insulin action on the vasculature and exit from the capillary beds; iv) insulin action in skeletal muscle, adipose tissue, liver, and central nervous system (CNS); v) final insulin degradation in the kidney [2]. Understanding such stages is clinically relevant, as defects in one or more of them can be associated with insulin resistance [2, 3, 4, 5]. In turn, insulin resistance is often associated with a cluster of cardiometabolic conditions (e.g., type 2 diabetes mellitus, obesity, atherosclerosis, and cardiovascular disease) that are collectively known as the metabolic syndrome (a.k.a. “insulin resistance syndrome”) and can result in high cardiovascular morbidity and mortality rates [6, 7]. Additionally, a thorough knowledge of the insulin bodily journey can assist clinicians in therapeutic decision-making for diabetic patients on exogenous insulin therapy in different clinical settings.

2. Insulin biosynthesis and release from pancreatic beta cells

Human insulin is a 51-amino acid peptide hormone synthesized and secreted by the pancreatic beta cells present within the islets of Langerhans. It consists of two polypeptide chains (A chain and B chain) linked together by two disulfide bonds (with an additional disulfide present within the A chain). The A chain consists of 21 amino acids, whereas the B chain consists of 30 amino acids. Insulin represents the paramount anabolic hormone promoting dietary carbon source deposition, with its major action sites being represented by the liver, skeletal muscle, and adipose tissue [2]. Production and secretion of insulin from pancreatic beta cells are crucial to maintaining normoglycemia. In humans, the insulin gene (INS) is located on chromosome 11. Its transcription is regulated by transcription factors (e.g., Pdx-1, NeuroD1, MafA) in response to increased circulating glucose levels [8]. Changes in glucose concentrations influence these beta-cell transcription factors at multiple levels, leading to parallel changes in their subcellular localization, expression levels, DNA-binding activity, transactivation capacity, and interaction with other proteins [8]. Once secreted, insulin exerts its functions by binding the insulin receptor (IR) expressed on target tissues. IR is a member of the ligand-activated receptor and tyrosine kinase family of transmembrane signaling proteins. The IR is a dimer of heterodimers that comprises two α-chains and two β-chains [9]. The human IR (hINSR) gene spans a region of greater than 120,000 base pairs (bp) on the short arm of chromosome 19 [10]. The Insulin Receptor Substrate (IRS) proteins represent a family of cytoplasmic adaptor proteins that transmit signals from the insulin and insulin-like growth factor 1 (IGF-1) receptors to evoke a cellular response [11].

In pancreatic beta cells, insulin is initially translated as preproinsulin, which is subsequently processed to proinsulin in the rough endoplasmic reticulum (RER) after the cleavage of its N-terminal signal peptide mediated by a signal peptidase. C-peptide (a.k.a. the connecting peptide) is a short 31-amino-acid peptide that connects insulin’s A-chain to its B-chain in the proinsulin molecule. In the RER, proinsulin undergoes rapid folding and disulfide bond formation. During this stage, the placement of C-peptide promotes the proper formation of disulfide bonds within and between the A and B peptide chains. After transit to the Golgi complex, proinsulin is sorted into immature secretory granules, where it is processed by prohormone convertases (PCSK1 and PCSK2) that act by cleaving the C-peptide. C-peptide is subsequently released into the bloodstream as a by-product of proinsulin proteolysis. Then, carboxypeptidase E (CPE) removes C-terminal basic amino acids from the resulting peptide chains, leading to the formation of mature insulin, a peptide hormone containing 51 amino acids distributed among the A and B chains of 21 and 30 amino acids, respectively. A and B chains are linked by two disulfide bonds, while another disulfide bond is formed within the A chain (Figure 1) [2, 12]. The zinc (Zn²⁺) cation co-crystallizes with insulin to form a hexameric crystal in the mature secretory granules. The zinc transporter 8 (ZnT8) or related zinc transporters have been suggested to mediate the transport of the insulin hexamer into the secretory granules [13]. Insulin granules dock with the plasma membrane through the coordinated interaction and recruitment of exocytic SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins [14]. Indeed, loss or altered activity of key SNARE proteins results in impaired insulin secretion [15].

Figure 1.

Physiologic stages of proinsulin processing in insulin-secreting pancreatic beta cells. Abbreviations: CPE, carboxypeptidase E; PCSK1, Proprotein convertase Subtilisin/Kexin type 1; PCSK2, Proprotein convertase Subtilisin/Kexin type 2; RER, rough endoplasmic reticulum. The figure was partly created with images adapted from Servier Medical Art licensed under a creative commons attribution 3.0 Unported license (https://smart.servier.com/).

Insulin synthesis is generally rapid (< 2 hours) and highly efficient, as only 1−2% of the protein remains as proinsulin within mature secretory granules. Hyperproinsulinemia indicates a pathological state arising from inefficient proinsulin processing within the beta-cell secretory granules and/or from premature release of proinsulin [16]. Indeed, the circulating proinsulin-to-C-peptide ratio (PI:C ratio) and proinsulin-to-insulin ratio (PIR) can be used as biomarkers of beta-cell endoplasmic reticulum dysfunction and inefficient proinsulin processing [17, 18, 19]. On the other hand, C-peptide is routinely used as a surrogate marker of endogenous insulin secretion due to the following reasons: C-peptide is secreted from pancreatic beta cells at an equimolar ratio to endogenous insulin, has negligible hepatic clearance, and is excreted at a more constant rate over a longer time compared to insulin, and its concentrations are not influenced by therapeutically administered exogenous insulin [20].

2.1 Biphasic insulin secretion

Glucose-stimulated insulin secretion (GSIS) in vitro consists of a transient first phase followed by a more sustained second phase. Shortly after the increase in glucose concentration following a meal, there is a transient stimulation of insulin secretion referred to as “first phase secretion.” This phase is then followed by a gradually developing secondary stimulation referred to as “second phase secretion” [21]. This biphasic insulin secretion reflects exocytosis of two distinct functional subsets of secretory granules in different pools [21]. Most granules (> 95%) belong to the reserve pool and are not available for release until they are physically translocated or chemically modified. The other subset of granules is referred to as the “readily releasable pool (RRP)” and contains less than 5% of the total granule number. Secretory granules proceed from the reserve pool into the RRP through a process called “mobilization,” which involves one or several ATP-dependent reactions [21, 22]. During the mobilization toward the cell periphery, granules must cross a cortical actin network to reach the plasma membrane. The rapid first phase of insulin secretion lasts up to 10 minutes and results from the fusion of the RRP granules with the plasma membrane. The RRP granules are already located at the membrane under basal conditions; these granules discharge their cargo in response to nutrient and non-nutrient secretagogues. The first phase of insulin secretion is then followed by a second phase associated with actin reorganization (to allow the recruitment of insulin secretory granules to the plasma membrane) and reaches a plateau at 2−3 hours [23]. Unlike the first phase, the second phase of insulin secretion can be evoked by nutrients and fuel secretagogues (like glucose) only and involves the mobilization of intracellular granules to t-SNARE sites at the plasma membrane to allow the distal docking and fusion steps of insulin exocytosis [24]. Thus, the actin cytoskeleton acts as a physical barrier to insulin secretion [24]. Conversely, the first phase secretion can also be elicited by non-metabolizable stimuli, suggesting that the second phase of insulin secretion represents an energy-dependent process. In type 2 diabetes mellitus (T2DM), there are alterations in the insulin release pattern that selectively involve the first phase of insulin secretion. This selective loss of the first phase insulin secretion can be the earliest detectable defect of beta-cell function after years of compensation for antecedent insulin resistance in subjects predisposed to develop T2DM. However, early abnormalities in the second phase of insulin secretion have also been described [21, 23].

2.2 Glucose sensing in pancreatic beta cells and insulin granule exocytosis

All the steps leading to the arrival, priming, docking, and fusion of secretory granules are regulated by a series of physiologic signals initiated by glucose, which represents the paramount metabolic signal evoking insulin secretion.

In humans, glucose enters the beta cell through glucose transporter type 1 (GLUT1) expressed on the cell membrane [25]. Afterward, the enzyme glucokinase (GK)—an isoform of hexokinase—rapidly phosphorylates glucose, leading to production of glucose-6-phosphate (G6P), which fuels the mitochondrial tricarboxylic acid (TCA) cycle via glycolysis and pyruvate generation. The completion of the TCA cycle leads to generation of ATP (adenosine triphosphate) from ADP (adenosine diphosphate) by ATP synthase, resulting in increases in the cytosolic ATP/ADP ratio. In turn, the increases in the cytosolic ATP/ADP ratio regulate cell membrane potential through inhibition and closure of ATP-sensitive K⁺ (K_ATP) channels, thus generating cell membrane depolarization (conversion of chemical to electrical signaling). When a certain threshold potential is exceeded (−55 mV to −50 mV), voltage-dependent Na⁺ and Ca²⁺ channels are activated and cause repetitive action potential spiking. The Ca²⁺ influx via voltage-gated Ca²⁺ channels results in the elevation of intracellular Ca²⁺ concentration, which subsequently triggers the fusion of the insulin granule bilayer with the plasma membrane (insulin granule exocytosis) and the release of insulin from the beta cell [26].

Although insulin is secreted primarily in response to glucose, additional nutrients and metabolic factors can amplify the glucose-induced insulin secretion, such as the gut-derived hormones called incretins (glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide), free fatty acids (FFAs), and amino acids [27]. Beta cells release insulin directly into the interstitial space of the pancreas, which is surrounded by a fenestrated endothelium that permits insulin to find its way into the portal circulation readily and to undergo the first-pass metabolism in the liver subsequently.

Beta cells’ electrical and Ca²⁺ responses within a pancreatic islet are synchronized thanks to gap junctions and paracrine and autocrine signals (e.g., ATP) [28, 29, 30]. The communication between beta cells within pancreatic islets contributes to the oscillations in insulin secretion, which occurs with a periodicity of 5−10 minutes in healthy individuals [31]. Yet, it is still unclear how several individual islets within the human pancreas (approximately one million) communicate and synchronize their insulin secretory oscillations to allow a pulsatile insulin release from the whole pancreas [2]. In this regard, it has been suggested that an intrapancreatic neural network may coordinate activity among different islet populations [2, 32].

3. First-pass hepatic insulin clearance and insulin action in the liver

Although pancreatic beta cells secrete insulin, the liver acts as a gatekeeper that regulates the amount of insulin allowed to reach the systemic circulation for action on target peripheral tissues through a process called “first-pass insulin clearance.” Therefore, the liver is the first organ that insulin encounters along its journey in the human body. It acts as a buffer to prevent peripheral hyperinsulinemia and subsequent insulin resistance in skeletal muscle and adipose tissue [6, 33]. The hepatic portal vein, which is the main vessel of the portal venous system, drains the blood from the gastrointestinal tract, gallbladder, pancreas, and spleen to the liver. As such, the portal vein also delivers insulin from the pancreas to the liver in discrete pulses occurring approximately every 5 minutes [34]. The magnitude of these insulin pulses varies between 0.5 and 1 nmol/L in the fasting state and ∼5 nmol/L after a meal [34, 35]. Enhanced insulin release in response to hyperglycemia is achieved by amplifying these high-frequency pulses [34]. The pulsatile insulin delivery through the portal vein to the liver regulates both hepatic insulin action and hepatic insulin extraction [36, 37].

Insulin reaches the liver through the portal vein at concentrations up to 10-fold higher than the insulin concentrations in the systemic circulation, thus creating a “portal-systemic gradient” mainly maintained by the significant hepatic insulin degradation [38]. Hence, the liver is exposed to substantially higher insulin concentrations than other insulin-responsive tissues such as skeletal muscle and adipose tissue.

Hepatocytes represent the cellular site where insulin clearance takes place. After its release into the portal circulation, insulin reaches the capillaries of the hepatic sinusoids, which, unlike other blood vessels, lack a basement membrane and are lined only by fenestrated (porous) endothelial cells [39]. The unique structure of hepatic sinusoids allows the exchange of contents between the blood and the surrounding hepatocytes. Thus, insulin easily diffuses from the portal circulation into the perisinusoidal space (the space between the sinusoids and the hepatocytes) and directly interacts with the hepatocyte surface. Receptor-mediated insulin uptake followed by insulin degradation in hepatocytes represents the underlying mechanism of hepatic insulin clearance [40]. Insulin binds the IR located on the microvilli of the hepatocyte membrane. The IR-insulin complex (receptor-ligand complex) is then internalized through clathrin-mediated endocytosis [41, 42, 43]. The subsequent loss of IR on the hepatocyte surface is followed by rapid recycling and reinsertion of intact and unbound IRs in the plasma membrane [44]. Michael et al. [45] showed that liver-specific insulin receptor knockout (LIRKO) mice exhibit dramatic insulin resistance due to alterations in the IR-mediated insulin endocytosis and degradation, which resulted in marked hyperinsulinemia due to a combination of increased insulin secretion and decreased insulin clearance. These findings support the importance of physiologic hepatic insulin signaling in regulating glucose homeostasis and maintaining normal liver function.

Hepatocytes express high levels of the enzyme CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a transmembrane glycoprotein phosphorylated by the IR and allowing the formation of an insulin-IR-CEACAM1 complex [46]. This complex upregulates the IR-mediated insulin endocytosis and degradation. The importance of CEACAM1 in hepatic insulin clearance has been demonstrated by animal studies showing that defects in insulin-stimulated hepatic CEACAM1 phosphorylation led to severe hyperinsulinemia and consequential insulin resistance, impaired glucose tolerance, and hyperglycemia [47]. The insulin degradation process begins upon insulin binding to the hepatocyte plasma membrane, where insulin is partly degraded by the extracellular insulin-degrading enzyme (IDE) [48]. The remaining insulin is degraded after internalization into the hepatocyte by IDE in endosomes and through proteolysis in lysosomes [2].

The half-life of insulin in the portal circulation is ~3−5 minutes [49]. Under physiologic conditions, up to 80% (usually 50%) of secreted insulin is degraded during its first-pass hepatic clearance [50]. Thus, the insulin concentration in the systemic circulation is about one-third that in the portal circulation (3:1 ratio of hepatic to peripheral insulin levels). The remaining undegraded insulin exits the liver through the hepatic veins and reaches the heart through the inferior vena cava. The heart then pumps insulin into the systemic (arterial) circulation toward the target tissues (skeletal muscle, adipose tissue, liver, and brain), where insulin exerts its actions. Approximately 25% of undegraded insulin returns to the liver (specifically, to the hepatic sinusoids) via the hepatic artery and undergoes a second round of insulin degradation in the hepatocytes (“second-pass hepatic insulin clearance”) [2].

Hepatic insulin degradation represents a physiologic mechanism modulating the amount of insulin that reaches the systemic circulation according to the metabolic demands and the insulin concentration that is required in the periphery. Under pathological conditions characterized by insulin resistance (e.g., obesity), there is a decreased hepatic insulin clearance rate that compensates for reduced peripheral insulin sensitivity and contributes (together with an increased insulin secretion rate) to the compensatory hyperinsulinemia of insulin-resistant individuals [51]. In this regard, Lee et al. showed that CEACAM1 expression is decreased in the hepatocytes of diabetic and nondiabetic patients with severe obesity and fatty liver disease, both of which are conditions commonly associated with insulin resistance [52].

3.1 Insulin action in the liver

Besides being involved in insulin degradation, the liver represents a major target tissue where insulin acts through the phosphoinositide 3-kinase (PI3K)/Akt pathway, which is a key signaling pathway mediating the insulin effects on anabolic metabolism in all organisms [3, 53]. Notably, insulin regulates hepatic glucose output by promoting glycolysis and glycogen synthesis (via activation of glycogen synthase) and by suppressing gluconeogenesis (de novo glucose production) and glycogenolysis (glycogen breakdown) [54]. This mechanism of action permits the hepatic storage of glucose as glycogen so that the hepatic glucose output prevails in the interprandial periods (when insulin action ceases) and/or upon stimulation by counterregulatory hormones (predominantly glucagon) that promote hepatic glycogenolysis and gluconeogenesis [55].

Glycogen synthesis (a.k.a. glycogenesis) and glycogenolysis are reciprocal pathways regulated by the balance between circulating insulin and glucagon concentrations under fed and fasting conditions, respectively [6]. Glucose production by the liver is finely regulated by insulin and counterregulatory hormones (e.g., glucagon), neural mechanisms, and nutrient supply. After glucose ingestion, insulin is released from the pancreatic beta cells into the portal circulation. It potently and rapidly inhibits hepatic glucose production (HPG) by directly suppressing glycogenolysis via the PI3K/Akt pathway, inhibiting the glycogen phosphorylase [6]. At this stage, insulin also promotes hepatic glucose uptake and glucose storage as glycogen by activating the enzyme glycogen synthase phosphatase, which catalyzes the dephosphorylation and subsequent activation of glycogen synthase [6, 56]. Moreover, insulin released from pancreatic beta cells after a meal suppresses HPG through its ability to inhibit the pancreatic alpha cell secretion of glucagon [57], a well-known counterregulatory hormone that stimulates hepatic gluconeogenesis [58].

Hepatic gluconeogenesis consists of the synthesis of glucose from non-carbohydrate precursors (such as lactate, pyruvate, glycerol, propionate, and alanine). It contributes to approximately 50% of all HGP following overnight fasting [6]. Although the liver is the major site of gluconeogenesis, a smaller contribution to this process is also provided by the kidney, small intestine, skeletal muscle, and brain [59, 60, 61, 62].

In the fed state, insulin inhibits hepatic gluconeogenesis primarily via direct mechanisms by acting as a potent negative regulator of different gluconeogenic enzymes in hepatocytes—such as glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK)—after its release into the portal circulation [6, 63, 64, 65]. However, insulin-mediated inhibition of hepatic gluconeogenesis also occurs through indirect mechanisms, namely via the insulin-mediated suppression of lipolysis, which results in decreased FFA availability and reduced hepatic acetyl-coenzyme A (acetyl-CoA) levels. Yet, this indirect pathway becomes relevant mainly when insulin is administered peripherally, and the physiologic (positive) portal-systemic blood insulin gradient is disrupted [6].

Under fasting conditions, glucagon inhibits the activity of the enzyme pyruvate kinase (PK), thus promoting the utilization of phosphoenolpyruvate for gluconeogenesis. In the fed state, insulin counteracts this process, favoring the uptake and oxidation of glucose in the liver [6, 66].

In the liver, insulin also controls lipid homeostasis by stimulating de novo lipogenesis and by regulating hepatic triacylglycerol (TAG) secretion via very-low-density lipoprotein (VLDL)-TAG export. This is in line with insulin acting as an anabolic hormone that promotes energy storage in both liver and adipose tissue. Specifically, insulin regulates VLDL production in the liver by targeting apolipoprotein B (apoB) for degradation and reducing apoB synthesis [67]. In the postprandial period, the increase in portal insulin concentrations reduces hepatic VLDL output and allows for temporary triglyceride storage for future secretion. Indeed, insulin induction of hepatic de novo lipogenesis favors triglyceride synthesis and occurs independently of insulin effects on apoB. Under conditions of insulin resistance, there is a higher apoB availability, which results in hepatic VLDL hypersecretion (coupled with stimulation of hepatic de novo lipogenesis). In turn, the hepatic VLDL hypersecretion results in the packaging of excess triglycerides into larger-sized VLDL particles (termed VLDL1) and more numerous VLDL particles, thus inducing hypertriglyceridemia [68]. Indeed, enhanced secretion of VLDL-TAG and hypertriglyceridemia are hallmarks of insulin-resistant conditions, such as obesity or non-alcoholic fatty liver disease (NAFLD) [53].

4. Insulin action in the vasculature

In systemic circulation, insulin starts to exert its hemodynamic effects on the vessels by promoting vasodilation and increasing blood flow to allow its own delivery to the peripheral tissues. In the macrovasculature, insulin binds the IR expressed on the endothelial cells and leads to the phosphorylation of the insulin receptor substrate 2 (IRS-2), which, in turn, results in the activation of class I PI3K that is linked downstream to the Akt/PKB (protein kinase B) signaling pathway. The downstream activation of Akt causes phosphorylation and activation of endothelial nitric oxide synthase (eNOS), which, in turn, catalyzes the synthesis of nitric oxide (NO) from L-arginine [69]. Subsequently, NO rapidly and freely diffuses to the adjacent outer layer of vascular smooth muscle cells (VSMCs), where it activates the intracellular enzyme guanylate cyclase that converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). The increase in intracellular cGMP concentration results in the activation of protein kinase G (PKG), which, in turn, causes reductions in intracellular Ca²⁺ concentration and subsequent relaxation of vascular smooth muscle and vasodilation via activation of the myosin light-chain phosphatase [70]. Insulin action in target tissues is temporally linked to the insulin’s vascular effects. For example, insulin acts on the vasculature to enhance its own delivery to skeletal muscle by inducing the relaxation of resistance vessels to increase blood flow and by promoting the relaxation of precapillary arterioles to increase the microvascular exchange surface perfused within the skeletal muscle (microvascular recruitment) [71]. Thus, insulin rapidly recruits skeletal muscle capillaries by a NO-dependent action, and the increase in capillary recruitment represents an early insulin effect promoting glucose uptake by the perfused myocytes [72]. Indeed, microvascular recruitment precedes both the activation of insulin signaling pathways and the increases in glucose disposal in skeletal muscle [72]. Under resting conditions, only 30−50% of muscle capillaries are perfused, and this proportion increases 2- to 3-fold in response to exercise and insulin action [6, 73]. During insulin resistance and in the presence of diabetes, obesity, and/or use of nitric oxide synthase inhibitors, vasoactive effects of insulin (including microvascular recruitment) can be impaired [71, 74, 75, 76].

When insulin reaches the capillaries of its target tissues (skeletal muscle and adipose tissue), it must pass through the capillary’s continuous endothelial lining to access the interstitial space and the parenchymal cells (myocytes and adipocytes). As capillaries are composed of a single layer of endothelial cells with interendothelial junctions, two possible routes have been proposed to explain the mechanism of insulin egress from the microvascular capillary endothelium toward the interstitial space and the parenchyma tissues (skeletal muscle and adipose tissue). In particular, insulin may cross the capillary endothelia via a transcellular route (through individual endothelial cells via receptor-mediated endocytosis and/or via fluid-phase endocytosis) or via a paracellular route (between adjacent endothelial cells) [2, 77].

It is worth noting that NO acts as a potent vasodilator and antiatherogenic molecule. NO synthesis is stimulated by insulin in VSMCs [78]. Thus, insulin exerts cardioprotective effects, whereas insulin resistance alters the NO synthesis causing accelerated atherosclerosis, coronary artery disease, and hypertension (all conditions frequently associated with obesity and/or T2DM). In addition, hyperinsulinemia secondary to insulin resistance leads to stimulation of the mitogen-activated protein kinase (MAPK) pathway, which results in enhanced VSMC growth and proliferation, inflammation, and atherosclerosis [79]. This occurs because MAPK can be activated either by insulin receptor substrate 1 (IRS-1) or by the adapter protein Shc; in insulin-resistant states, phosphorylation of IRS-1 on tyrosine residue is impaired, while Shc activation remains intact [6].

5. Insulin action in skeletal muscle and adipose tissue

Insulin acts as the master regulator of glucose metabolism, with one of its major actions being the increase in glucose uptake by skeletal muscle and adipose tissue. This process involves the translocation of the polypeptide called glucose transporter type 4 (GLUT4) into the plasma membrane of myocytes and adipocytes. Within minutes of insulin binding to its receptor, cytoplasmic vesicles containing GLUT4 (constituting a specialized intracellular organelle called “GLUT4 storage vesicles” or GSVs) translocate from the cytoplasm to the cell surface, fuse their membrane with the plasma membrane, and insert GLUT4 glucose transporters into the plasma membrane, thus permitting glucose uptake [80]. GLUT4 translocation is finely regulated at several stages, and this process does not involve the internalization of the insulin-IR complex.

The GLUT4 storage compartment is in dynamic communication with general recycling endosomes, thus generating a steady-state whereby ∼90−95% of GLUT4 resides intracellularly at any point in time in both adipose and muscle cells [2, 81]. In muscle and adipose cells, insulin binds to IR on the cell surface, promotes the IR tyrosine kinase activity toward autophosphorylation, and activates the downstream insulin signaling cascade to PI3K and Akt, which results in the dynamic remodeling of cortical actin filaments and rapid mobilization of GSVs from perinuclear/cytosolic depots toward the cell periphery; this mobilization ultimately allows GSV tethering, docking, and fusion with the plasma membrane [2, 82]. Reduced GLUT4 translocation to the plasma membrane has been described in skeletal muscle of diabetic rodents and humans as a consequence of defects in the insulin-mediated signals promoting GLUT4 vesicle release from the GSV organelle and GLUT4 vesicle interaction-fusion with the plasma membrane [83, 84].

The GLUT4 transporter is expressed in all insulin-sensitive tissues, including adipose tissue and skeletal and cardiac muscle [6, 85]. On the other hand, GLUT1 is insulin-insensitive and represents the basal glucose transporter in adipose tissue, skeletal muscle, erythrocytes, and the brain. Glucose transporter type 2 (GLUT2) is the major glucose transporter required for glucose sensing and hepatic glucose uptake and output; it is the major member of the GLUT family in pancreatic beta cells and hepatocytes, but it is also abundant in the intestine and kidney [6, 86].

Approximately 60−70% of the ingested glucose load escapes the hepatic glucose uptake and is disposed of (under the insulin action) by muscle and other obligate glucose utilizers such as smooth muscle, CNS, and formed blood elements, with about two-thirds of the glucose entering the glycogen synthetic pathway and about one-third entering the glycolytic pathway [6, 87]. Quantitatively, the glucose uptake by adipose tissue accounts for about 5−10% of the ingested glucose load [6].

5.1 Insulin action in skeletal muscle

Skeletal muscle represents the body’s largest reservoir of carbohydrate and protein. Therefore, it is highly dependent on the insulin-mediated uptake of glucose and amino acids to sustain such intracellular nutrient pools [6]. Following a meal, approximately 60−70% of the ingested carbohydrate is disposed of (under the insulin action) by skeletal muscle, highlighting the critical role of insulin-mediated muscle glucose uptake in postprandial glycemic control [6, 88].

In skeletal muscle, insulin binds to the extracellular domain (α subunits) of the IR expressed on the myocellular membrane. This binding leads to the transphosphorylation of the intracellular IR β subunits and to the tyrosine phosphorylation of IRS proteins [6, 89, 90]. Then, phosphorylated IRS-1 activates PI3K, leading to the generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and subsequent phosphorylation and activation of Akt, that ultimately stimulates GLUT4 translocation from the intracellular GSV organelle to the plasma membrane [6, 91].

In myocytes, insulin upregulates the transcription and activity of the enzyme hexokinase II. This enzyme catalyzes the intracellular phosphorylation of glucose to G6P [92, 93, 94], which is readily utilized for the synthesis and storage of glycogen and conversion into pyruvate via the glycolytic pathway. Insulin activates the enzyme pyruvate dehydrogenase, which transforms pyruvate into acetyl-CoA. Skeletal muscle glycolysis is primarily driven by the rate of acetyl-CoA generation in the mitochondria [6, 95]. The increase in intracellular concentration of G6P also promotes glycogen synthesis (especially in the presence of sustained muscle glucose uptake), since G6P acts as a major allosteric activator of glycogen synthesis via inhibition of glycogen phosphorylase and activation of glycogen synthase [96, 97]. Impairment in insulin-induced hexokinase II expression has been suggested to contribute to insulin resistance in obesity and T2DM [98].

According to its anabolic actions, insulin plays an essential role in the regulation of muscle protein metabolism and maintenance of skeletal muscle mass by suppressing muscle proteolysis (through the inactivation of autophagy mediated by Akt) and by stimulating muscle protein synthesis (via activation of the mammalian target of rapamycin complex 1, a key effector of cellular protein synthesis) [6, 99]. After ingesting a meal, the increase in circulating amino acid concentrations allows for the net stimulation of muscle protein synthesis induced by the postprandial rise in circulating insulin levels [6, 100].

5.2 Insulin action in adipose tissue

Adipose tissue represents the major storage site of fatty acids, which are released in the circulation in the form of FFAs after the process called “lipolysis”, which consists of the hydrolysis of triglycerides within the adipocytes. During fasting conditions, lipolysis allows for the use of FFAs as an energy source for peripheral tissues. Insulin plays a crucial role in adipose tissue metabolism by stimulating glucose uptake, inhibiting lipolysis (in a dose-response manner), and promoting lipogenesis (with subsequent storage of triglycerides in adipocytes) [6]. FFAs are transported into adipocytes by the scavenger receptor CD36, while glucose is transported into adipocytes by GLUT1 (insulin-independent) and GLUT-4 (insulin-dependent) transporters. The intracellular glucose is then used to synthesize glycerol-3-phosphate (G3P), which is necessary for triglyceride synthesis. Indeed, triglyceride synthesis starts with the generation of diacylglycerols (DAG) from the conversion of glucose into G3P and the binding of two fatty acid acyl-CoA molecules [6].

It has been shown that GLUT4 expression is reduced in insulin-resistant patients with obesity and T2DM [101], thus explaining the reduced glucose uptake observed in visceral and subcutaneous fat of insulin-resistant subjects [6].

In adipocytes, insulin binds to IR leading to tyrosine phosphorylation and activation of the IR, resulting in the interaction with IRS-1 and IRS-2 and subsequent activation of the PI3K complex [6]. Insulin-mediated activation of PKB promotes the activation of the mammalian target of rapamycin complex 1 (mTORC1), which ultimately results in the inhibition of the adipose triglyceride lipase (ATGL). The enzyme ATGL stimulates the hydrolysis of triglycerides to DAG, with subsequent release of one fatty acid molecule. Insulin also inhibits, via a PKB/Akt-dependent action, the subsequent steps of lipolysis by reducing the activity of the enzyme hormone-sensitive lipase (HSL), which hydrolyzes DAG to monoacylglycerol (MAG) with the release of one fatty acid molecule [6, 102].

In patients with obesity and/or insulin resistance, a resistance to the antilipolytic effect of insulin leads to a chronic increase in circulating levels of FFAs under fasting and fed conditions (despite the postprandial hyperinsulinemia). The chronic increase in circulating FFA levels causes detrimental effects via lipid accumulation in non-adipose tissues such as liver, skeletal muscle, heart, kidney and pancreatic beta cells (a phenomenon known as “lipotoxicity”), and potentially leads to the development of T2DM [103, 104, 105].

6. Insulin action in the brain

The brain relies on glucose as its major fuel. Glucose is transported across the cell membranes by facilitated diffusion mediated by two main glucose transporter proteins (GLUT1 and GLUT3) that are differentially expressed in brain endothelial cells, astrocytes, and neurons [106]. The glucose transport into most neurons depends on glucose transporter type 3 (GLUT3). In contrast, the glia and brain endothelial cells depend on GLUT1 activity for glucose uptake from brain interstitial fluid and plasma, respectively [107]. As insulin is not needed for GLUT1- or GLUT3-mediated glucose transport, it is not required for glucose transport into most brain cells [108]. Therefore, the brain and the CNS have long been considered “insulin insensitive and unresponsive.” However, evidence from the last two decades has supported that the brain is an insulin-responsive organ and that insulin exerts neuroregulatory properties and additional pleiotropic actions in the CNS [109]. First, insulin can cross the blood-brain barrier via a receptor-mediated process [110], thus achieving concentrations in the cerebrospinal fluid that are approximately one-third of those in the systemic circulation. In the CNS, insulin primarily regulates appetite and energy expenditure by decreasing the expression of the two orexigenic neuropeptides, namely neuropeptide Y (NPY) and agouti-related peptide (AGRP)—released by the same set of neurons within the arcuate nucleus of the hypothalamus—and by increasing the expression of the anorexigenic polypeptide pro-opiomelanocortin (POMC) [111, 112, 113].

Growing evidence over the past decade has also shed light on insulin’s function in the brain as an important regulator of mood, memory, and cognition, in light of the trophic and developmental actions of insulin on neurons and glial cells. In this regard, accumulating evidence suggests that intranasal insulin administration may represent a valid therapeutic option to improve cognitive function in neurodegenerative diseases by means of its ability to increase cerebral insulin levels and reverse brain insulin resistance [114, 115, 116, 117]. Indeed, defects in brain insulin action and altered insulin receptor signaling have emerged as potential contributors to Alzheimer’s disease pathophysiology, so this condition has also been termed “type 3 diabetes” [118, 119]. Defects in insulin signaling also provide a link between diabetes and CNS disorders through direct neuronal effects and regulation of mitochondrial function, cholesterol synthesis, tau phosphorylation, and amyloid-beta peptide (Aβ) processing [117, 120].

In the brain, insulin also influences body temperature control, since insulin or IGF-1 injection into the preoptic area of the hypothalamus can activate brown adipose tissue and induce hyperthermia. This effect is lost in neuron-specific IR knockout (NIRKO) mice, suggesting that it is mediated by IR [121]. Studies in NIRKO mice have also shown that insulin acts in the brain to regulate the counterregulatory response to hypoglycemia by directly altering glucose sensing in hypothalamic neurons and shifting the glycemic levels necessary to evoke a normal sympathoadrenal response [122]. Yet, the exact cellular mechanisms underlying all the abovementioned actions of insulin in the CNS remain to be elucidated.

7. Brief overview of systemic insulin actions following postprandial insulin secretion

Insulin is the paramount anabolic hormone promoting carbon energy storage in adipose tissue, skeletal muscle, and liver. As such, insulin represents the master regulator of glucose, lipid, and protein metabolism. Following glucose ingestion, there is a rise in plasma glucose concentrations and subsequent glucose-mediated stimulation of insulin secretion by pancreatic beta cells. The resultant hyperglycemia and hyperinsulinemia determine a series of closely coordinated metabolic responses that return the plasma glucose concentration to normal values within 2 hours, namely: a) inhibition of glucagon secretion from pancreatic alpha cells and direct suppression of endogenous glucose production (mainly HGP); b) inhibition of lipolysis and subsequent decline in plasma FFA and glycerol concentrations, which results in the indirect suppression of HGP and augmented muscle glucose uptake (as FFAs promote HGP and inhibit muscle glucose uptake) [123]; c) stimulation of glucose uptake by myocytes, hepatocytes, and adipocytes; d) skeletal muscle vasodilation contributing to enhanced muscle glucose disposal [6].

8. The role of the kidney in insulin degradation and excretion

Kidneys represent the final “station” of the insulin journey, since they are involved in the last steps of degradation and excretion of the remaining circulating insulin that has not previously been degraded by the liver and that has exerted its peripheral actions. Insulin has a short plasma half-life (about 4−6 minutes), as it is expected from the need to respond rapidly to changes in blood glucose levels [124, 125]. Insulin uptake and degradation represent features of all insulin-sensitive tissues, where the hormone is slowly internalized by cells (such as adipocytes and myocytes) and is then transported to lysosomes for degradation [2]. As it has previously been mentioned, the liver is the primary site of insulin clearance, where approximately 50% of insulin is degraded in the first-pass hepatic clearance and 25% of insulin is degraded in the second-pass hepatic clearance. The clearance of the remaining 25% of undegraded insulin initially released from the pancreas occurs in the kidneys.

Insulin clearance by the kidney occurs via two main mechanisms: i) glomerular filtration and ii) proximal tubular reabsorption and degradation [50, 126]. Glomerular clearance of insulin occurs through both nonspecific diffusion and specific receptor-mediated transport. Thus, insulin is freely filtered across the capillaries of the glomerulus (given its molecular weight of 5808 Da), enters the luminal space, and reaches the proximal tubule. Then, more than 99% of the filtered insulin is reabsorbed by proximal tabular epithelial cells mainly via endocytosis (possibly mediated by scavenger receptors such as megalin) [127]. In contrast, less than 1% of filtered insulin appears in the final urine [2, 50]. After being internalized into the proximal tabular epithelial cells, insulin dissociates from its binding sites and proceeds to lysosomes for degradation. Renal insulin clearance also occurs through tubular secretory clearance from post-glomerular, peritubular capillaries via receptor-mediated processes. In particular, IRs expressed on the contraluminal membrane of the epithelial cells (particularly those lining the distal convoluted tubule of the nephron) bind insulin (that comes via diffusion from peritubular capillaries) and transport the hormone intracellularly for degradation [128, 129].

Renal clearance of insulin amounts to approximately 200 mL/min, significantly exceeding the normal glomerular filtration rate (GFR) of 120 mL/min because of the contribution of tubular secretion [130]. It has been proposed that 6 to 8 units of insulin are degraded by the kidneys each day via the two abovementioned mechanisms; this amount accounts for about 25% of the daily pancreatic insulin production [130]. In diabetic patients treated with insulin therapy, the contribution of renal insulin metabolism is even higher than that of non-diabetic subjects or non-insulin-treated diabetic patients, as exogenous insulin enters the systemic circulation directly, without undergoing the first-pass metabolism in the liver. On the other hand, advanced chronic kidney disease (as can be observed in diabetic nephropathy) unavoidably alters the renal insulin clearance and prolongs the half-life of circulating insulin (although a certain degree of insulin resistance may also be present in the early stages of chronic kidney disease) [131]. This often leads to a substantial reduction in the daily insulin requirements of diabetic patients treated with exogenous insulin [131], thus highlighting the importance of renal insulin clearance as a mechanism to regulate insulin’s plasma half-life and end the insulin action.

Besides playing a pivotal role in the clearance and degradation of circulating insulin, the kidneys also represent an important site of insulin action. Indeed, IRs expressed on the renal tubular epithelial cells act as sites sensing insulin and promoting important insulin functions, such as inhibition of renal gluconeogenesis, upregulation of renal sodium-glucose co-transporter 2 (SGLT2) protein expression, and reabsorption of sodium, phosphate, and glucose [129, 132]. As it has previously been mentioned, kidneys also contribute to gluconeogenesis, although to a lesser extent compared to the liver. It is now established that, under postabsorptive conditions, 80−90% of the basal rate of endogenous glucose production comes from the liver, with the remaining 10−20% coming from the kidneys [6, 133]. Accordingly, potential factors that explain the increased risk of hypoglycemia observed in chronic kidney disease include reduced renal insulin clearance, decreased caloric intake, impaired release of the counterregulatory hormone epinephrine (due to autonomic neuropathy), concurrent liver failure, reduced metabolism of substances and drugs that can cause a reduction in plasma glucose levels (e.g., alcohol, nonselective beta-blockers, disopyramide), and reduced renal gluconeogenesis [131, 134]. Similar to its action in the liver, insulin suppresses renal gluconeogenesis [135]. However, insulin-resistant states (like T2DM) can cause an impairment of insulin actions in the kidney, leading to increased renal gluconeogenesis [133]. Figure 2 illustrates the insulin journey in the human body across the “insulin highway.”

Figure 2.

The insulin journey in the human body across the “insulin highway.” Abbreviations: CNS, central nervous system. The figure was conceived by Marco Infante and was partly created with images adapted from Servier Medical Art licensed under a creative commons attribution 3.0 Unported license (https://smart.servier.com/). Digital figure drawing by Enzo Luchetti.

9. Conclusions

Insulin is the paramount anabolic hormone acting as the master regulator of glucose, lipid, and protein metabolism. During fasting and fed states, insulin regulates the flux of nutrients such as glucose, FFAs and amino acids, between liver, skeletal muscle and adipose tissue to ensure a proper nutrient supply to cells and maintain normal glucose homeostasis. Understanding the sequential stages of the physiologic journey of insulin in the human body is pivotal to comprehending the clinical consequences of derangements of insulin secretion and action. Indeed, defects in one or more of such stages can be associated with the development of insulin resistance, metabolic syndrome, and T2DM. Moreover, a thorough knowledge of the insulin bodily journey can assist clinicians in therapeutic decision-making for diabetic patients on exogenous insulin therapy in different clinical settings. The abovementioned stages include the following: i) insulin synthesis and release from pancreatic beta cells; ii) insulin first-pass metabolism and partial clearance in the liver; iii) insulin action on the vasculature and exit from the capillary beds; iv) insulin action in peripheral and central target tissues (skeletal muscle, adipose tissue, liver, and CNS); v) final insulin degradation in the kidney. Each of these stages is finely regulated by complex intracellular mechanisms that take place in different tissues and allow for the anabolic actions of insulin. Yet, several cellular and molecular aspects of the insulin bodily journey are still not entirely clear and need to be elucidated.

Acknowledgments

I would like to thank Mr. Enzo Luchetti for his thorough work in creating the digital figure drawing (Figure 2).

Conflict of interest

The author declares no conflict of interest.