3.1. Paracrine Regulation

Omar-Hmeadi et al. observed a lack of inhibition in glucagon exocytosis by hyperglycaemia, somatostatin, or insulin in intact islets in α cells from T2DM cadaveric. Instead, hyperglycaemia inhibits α cell exocytosis, but not in the T2DM donor’s α cell or when paracrine inhibition by insulin or somatostatin is blocked. A reduced Surface expression of Somatostatin-receptor-2 in islet from T2DM donors suggests somatostatin resistance, and consequently, elevated glucagon in T2DM may reflect α cell insensitivity to paracrine inhibition during hyperglycaemia ^[12][16].

3.2. Autocrine Regulation

It is widely known that α cell membranes contain a large number of glucagon receptors (GR) ^[13][32]. Furthermore, a recent study reported that GR activation increases glucagon gene transcription via cAMP response element-binding (CREB) activation by the PKA-dependent pathway ^[14][40]. Moreover, several studies have reported that blocking GRs has been reported to improve glucose homeostasis ^[15][16][17][41,42,43], due to glucagon stimulates its own release acting on GR on α cells ^[18][44]. In fact, in a study conducted by Liu et al. on αTC1 cells (a pancreatic alpha cell line derived from an adenoma created in transgenic mice), immunofluorescent staining confirmed the presence of GRs on αTC1 cells. After 72 h of treatment with GRs antagonist (to block the effects of the endogenous glucagon), a 44% decrease in αTC1 cell proliferation was observed compared with the control group by counting the cells in the S phase. These results show that glucagon has direct trophic effects on α cells by an autocrine mechanism, and when the pancreatic α-cell number was decreased in db/db mice by a glucagon receptor antagonist, plasma glucagon levels were significantly decreased too ^[19][45].

3.3. Juxtacrine Regulation

The juxtacrine mechanism is a common type of signalling between adjacent cells requiring direct contact between cells. Recently, a juxtacrine connection between α- and β-cells has been documented as another exciting way in glucagon secretion control since the confirmation of Eph/ephrin system between α- and β cells ^[20][21][22][51,52,53]. The Eph signalling system belongs to the superfamily of transmembrane Tyr kinase receptors. Today, it is well established that Eph allows short-distance cell-cell interaction by binding with their specific ligands (ephrin), primarily affecting cytoskeleton and leading to cell repulsion or adhesion in some circumstances. In this regard, the first studies in this field showed that many processes involving fast changes in cellular morphology were ephrin–Eph dependent ^[23][54]. More recently, other critical ephrin–Eph signalling-mediated processes have been identified and characterised, e.g., axon guidance, synaptic plasticity, cancer, and processes like juxtacrine hormones release control involving short-distance cell-cell communication. In addition, there is increasing evidence about its influence on cell differentiation, proliferation, and apoptosis regulation ^[24][55].

3.4. Endocrine Regulation

Non-pancreatic hormones also have an active role in α cell regulation. For example, Bagger et al. ^[25][66] demonstrated that intravenous (IV) glucose administration did not reduce glucagon levels in subjects with T2DM; however, it was achieved with oral glucose administration, suggesting a potential role of intestine lining and incretins on α cell. In addition, GLP-1 and gastric inhibitory polypeptide (GIP) are hormones encoded by the GCG gene (the same encodes glucagon), secreted by enterocytes, with well-studied insulinotropic effects ^[26][67]. However, considering the difficulty of these experimental studies, its activity on α cells remains disputed ^[26][27][67,68]. The regulation of glucagon secretion relies on both intrinsic mechanisms in the α cell and extrinsic mechanisms in neighbouring and nearby cells since even autacoids like serotonin exhibit receptors in the α cell membrane inhibiting glucagon release. When α cells have a weak serotonergic tone, they lose their glucose-dependent inhibition of the glucagon-release, generating hyperglucagonemia under hyperglycaemic conditions.

4. Hyperglucagonaemia and α Cell Dysfunction in Diabetes

Extensive emerging evidence has been published among the “bi-hormonal theory” in T2DM pathogenesis in which the coexistence of hyperglucagonemia and relative insulin deficiency increase gluconeogenesis and exacerbates peripheral IR ^[28][74], leading to overt T2DM development. Currently, there is some consensus regarding hyperglucagonemia origin. This is centred on two possible mechanisms: (1) a progressive loss in the regulatory mechanisms in the secretion patterns due to α cells functional alterations ^[29][30][75,76], or (2) modification in both the islet microarchitecture and cellularity ^{[31][32][33][34]}[77,78,79,80].

4.1. Structural Alterations in Pancreatic Islet

Conclusive evidence has demonstrated a β cell mass reduction and a concomitant decrease in insulin secretion in subjects with long-standing T2DM ^[35][36][62,63] and a sensible fall in GABA and serotonin release, which are crucial paracrine regulators of glucagon secretion, as explained previously. However, post-mortem studies have reported an increased α cell mass in subjects with diabetes. Nonetheless, the evidence is not conclusive ^[36][63]. This finding can be related to a loss of α cell regulating factors or a compensatory mechanism secondary to β cells mass loss ^[36][63]. Nevertheless, multiple studies have reported that an elevation of Interleukin 6 (IL-6) circulating levels in T2DM adult mice is possibly linked with an expansion of α cell mass and hyperglucagonemia ^[37][64], suggesting that α cell proliferation in Type 1 Diabetes Mellitus (T1DM) is probably IL-6-dependent ^[38][81].

4.2. Alpha Pancreatic Cell Dysfunction, Energetic Sensors, and Ionic Channels

Dysfunction in glucose-mediated glucagon releasing mechanisms in α cells is extensively assessed in T2DM ^[39][106]. In this regard, Zhang et al. ^[40][107], using islets from T2DM organ donors, reported that glucose did not inhibit glucagon secretion. Furthermore, they replicated this methodology in non-diabetic samples by administering diazoxide, a well-known K_ATP opener, and oligomycin as an inhibitor of ATP production. Interestingly, in pancreatic islets treated with oligomycin, tolbutamide administration restored glucagon secretion, suggesting that DM hyperglucagonemia is linked to an impaired α cell K_ATP inhibition ^[40][107]. A recent in vivo study by Knudsen et al. ^[41][108] performed on mice lacking fumarase (Fh1) in their β and α cells developed spontaneous T2DM over 10 weeks of age secondary to an impairment of insulin secretion and found a lack of glucagon inhibition on hyper- glycaemic mice. They proposed that hyperglycaemia causes an increment of intracellular sodium in α-cell, secondary to the overactivity of SGLT in these cells. To overcome this, sodium antiporters, as Sodium-H+ transporters, are activated to reduce cellular sodium, conducing to significant cytoplasmic acidification. A subsequent reduction of mitochondrial matrix Ph occurred due to changes in cell electrochemical gradient, carrying an inhibition of oxidative phosphorylation, a decrease of ATP production, a decrease in ATP/ADP ratio, and a consequent opening of K_ATP, which culminate with an inappropriate glucagon secretion during a hyperglycaemic state. The researchers also hypothesised that mitochondrial and cytoplasmic acidification would lead to a reduction in fumarase activity ^[41][42][108,109], leading to an accumulation of fumarate in the cytoplasm. Thus, glycolysis may contribute to the worsening of the chaotic state of acidification and K_ATP activation in α cell during hyperglycaemia. Knudsen et al. ^[41][108] also set the inhibition of fumarase and disruption of ATP production may be found in other SGLT-expressing cells. It might be related, in part, to the kidney and cardiac failure observed in diabetes, which is also one of the plausible explanations for the cardiovascular risk and renal benefits of SLGT-1 inhibitors in T2DM patients ^[43][44][110,111]. Impaired Glucokinase (GCK) activity is another metabolic alteration linked to hyperglucagonemia in diabetes. Basco et al. ^[45][112] developed an in vivo study on GCK-deficient mice reporting an increase in post-prandial glucagon levels with hyper-glucagonemia, an increment in the liver gluconeogenic gene expression, and therefore, a hepatic glucose output increment. This fact may point to evidence of an underexpression/dysfunction of α-cell-GCK in T2DM, suggesting that GCK enhancers are a promissory therapy, not merely for their effects on insulin but also for their inhibitory effects on glucagon secretion. However, a recent trial with a novel GCK activator, HMS5552, did not report any significant improvement in glucagon levels ^[46][113]. Therefore, islet alterations (such as reduced β-cell mass) and α-cell alterations are typical phenomena of long-standing T2DM and possible transformation into undifferentiated cell forms or other islet cells through glucotoxicity. Otherwise, α cell K_ATP impairment is hypothesised as a hyperglucagonemia helper and lacks fumarase activity in DM.