Summary Early in the pathogenesis of Type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α-cells occurs prior to impaired glucose stimulated insulin secretion (GSIS) from β-cells. However, whether hyperglucagonemia is causally linked to β-cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on β-cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose intolerant high fat diet fed and Leprdb/db mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

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Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2′-deoxyuridine incorporation, [3H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP−/− mice, in INS-1–derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells.

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced β-cell mass (11, 62). Therapies aimed at expanding β-cell mass may be useful to treat T2D (14). Although feeding rodents a high-fat diet (HFD) for an extended period (3-6 mo) increases β-cell mass by inducing β-cell proliferation (16, 20, 53, 54), evidence suggests that adult human β-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human β-cells, are not known. To develop a model to identify early drivers of β-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The β-cell proliferative response also began within the first week of HFD feeding. Intriguingly, β-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory β-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse β-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human β-cell proliferation.

An important goal in diabetes research is to understand the processes that trigger endogenous β-cell proliferation. Hyperglycemia induces β-cell replication, but the mechanism remains debated. A prime candidate is insulin, which acts locally through the insulin receptor. Having previously developed an in vivo mouse hyperglycemia model, we tested whether glucose induces β-cell proliferation through insulin signaling. By using mice lacking insulin signaling intermediate insulin receptor substrate 2 (IRS2), we confirmed that hyperglycemia-induced β-cell proliferation requires IRS2 both in vivo and ex vivo. Of note, insulin receptor activation was not required for glucose-induced proliferation, and insulin itself was not sufficient to drive replication. Glucose and insulin caused similar acute signaling in mouse islets, but chronic signaling differed markedly, with mammalian target of rapamycin (MTOR) and extracellular signal–related kinase (ERK) activation by glucose and AKT activation by insulin. MTOR but not ERK activation was required for glucose-induced proliferation. Cyclin D2 was necessary for glucose-induced β-cell proliferation. Cyclin D2 expression was reduced when either IRS2 or MTOR signaling was lost, and restoring cyclin D2 expression rescued the proliferation defect. Human islets shared many of these regulatory pathways. Taken together, these results support a model in which IRS2, MTOR, and cyclin D2, but not the insulin receptor, mediate glucose-induced proliferation.

Pancreatic β-cell proliferation is infrequent in adult humans and is not increased in type 2 diabetes despite obesity and insulin resistance, suggesting the existence of inhibitory factors. Free fatty acids (FFAs) may influence proliferation. In order to test whether FFAs restrict β-cell proliferation in vivo, mice were intravenously infused with saline, Liposyn II, glucose, or both, continuously for 4 days. Lipid infusion did not alter basal β-cell proliferation, but blocked glucose-stimulated proliferation, without inducing excess β-cell death. In vitro exposure to FFAs inhibited proliferation in both primary mouse β-cells and in rat insulinoma (INS-1) cells, indicating a direct effect on β-cells. Two of the fatty acids present in Liposyn II, linoleic acid and palmitic acid, both reduced proliferation. FFAs did not interfere with cyclin D2 induction or nuclear localization by glucose, but increased expression of inhibitor of cyclin dependent kinase 4 (INK4) family cell cycle inhibitors p16 and p18. Knockdown of either p16 or p18 rescued the antiproliferative effect of FFAs. These data provide evidence for a novel antiproliferative form of β-cell glucolipotoxicity: FFAs restrain glucose-stimulated β-cell proliferation in vivo and in vitro through cell cycle inhibitors p16 and p18. If FFAs reduce proliferation induced by obesity and insulin resistance, targeting this pathway may lead to new treatment approaches to prevent diabetes.

Adaptive β-cell replication occurs in response to increased metabolic demand during insulin resistance. The intracellular mediators of this compensatory response are poorly defined and their identification could provide significant targets for β-cell regeneration therapies. Here we show that glucose and insulin in vitro and insulin resistance in vivo activate protein kinase C ζ (PKCζ) in pancreatic islets and β-cells. PKCζ is required for glucose- and glucokinase activator–induced proliferation of rodent and human β-cells in vitro. Furthermore, either kinase-dead PKCζ expression (KD-PKCζ) or disruption of PKCζ in mouse β-cells blocks compensatory β-cell replication when acute hyperglycemia/hyperinsulinemia is induced. Importantly, KD-PKCζ inhibits insulin resistance–mediated mammalian target of rapamycin (mTOR) activation and cyclin-D2 upregulation independent of Akt activation. In summary, PKCζ activation is key for early compensatory β-cell replication in insulin resistance by regulating the downstream signals mTOR and cyclin-D2. This suggests that alterations in PKCζ expression or activity might contribute to inadequate β-cell mass expansion and β-cell failure leading to type 2 diabetes.

Genome-wide association studies link the locus with type 2 diabetes (T2D) risk, but mechanisms increasing risk remain unknown. The locus encodes cell cycle inhibitors ,, and ;; and , a long noncoding RNA. The goal of this study was to determine whether T2D risk SNPs impact locus gene expression, insulin secretion, or β-cell proliferation in human islets. Islets from donors without diabetes ( = 95) were tested for SNP genotype (rs10811661, rs2383208, rs564398, and rs10757283), gene expression (, ,, ,, ,, and ), insulin secretion ( = 61), and β-cell proliferation ( = 47). Intriguingly, locus genes were coregulated in islets in two physically overlapping cassettes: -, which increased with age, and -, which did not. Risk alleles at rs10811661 and rs2383208 were differentially associated with expression of , but not, , or, in age-dependent fashion, such that younger homozygous risk donors had higher expression, equivalent to older donor levels. We identified several risk SNP combinations that may impact locus gene expression, suggesting possible mechanisms by which SNPs impact locus biology. Risk allele carriers at coding SNP rs564398 had reduced β-cell proliferation index. In conclusion, locus SNPs may impact T2D risk by modulating islet gene expression and β-cell proliferation.