Recently, it was reported that angiopoietin-like protein 8 (ANGPTL8) was the long-sought “betatrophin” that could control pancreatic beta cell proliferation. However, studies of Angptl8 −/− mice revealed profound reduction of triglyceride levels, but no abnormalities in glucose homeostasis. We now report that Angptl8 −/− mice undergo entirely normal beta cell expansion in response to insulin resistance resulting from either a high-fat diet or from the administration of the insulin receptor antagonist S961. Furthermore, overexpression of ANGPTL8 in livers of mice doubles plasma triglyceride levels, but does not alter beta cell expansion nor glucose metabolism. These data indicate that ANGPTL8 does not play a role in controlling beta cell growth, nor can it be given to induce such expansion. The findings that plasma triglyceride levels are reduced by Angptl8 deletion and increased following ANGPTL8 overexpression support the possibility that inhibition of ANGPTL8 represents a therapeutic strategy for hypertriglyceridemia.

These findings appear to be in conflict with the observation that Angptl8mice fed either a chow or high-fat diet have no alteration in glucose homeostasis (). In this study, we used Angptl8mice to determine if ANGPTL8 is required for beta cell expansion in insulin resistant states as had been previously reported by

Angiopoietin-like protein 8 (ANGPTL8) is a circulating protein that is expressed primarily in liver and adipose tissue. Hepatic overexpression of ANGPTL8 in mice is associated with hypertriglyceridemia, whereas inactivation of Angptl8 causes a reduction in plasma triglyceride levels (). ANGPTL8 was recently reported to mediate an increase in beta cell proliferation and beta cell mass in mice where insulin resistance was induced by the insulin receptor antagonist S961 (). Overexpression of ANGPTL8, which the authors refer to as betatrophin, induced beta cell proliferation, expansion of beta cell mass, and led to improved glycemic control ().

The pancreatic beta cell possesses the capacity to self-duplicate (). Insulin resistance elicits a compensatory increase in insulin secretion to maintain normoglycemia. Type 2 diabetes develops when the beta cells cannot compensate for the increase in insulin requirement. After an initial compensatory expansion, beta cell function and mass progressively declines (). The factors that expand beta cell mass in response to insulin resistance have not been molecularly defined.

Infusion of the insulin receptor antagonist S961 (20 nMol/week) caused a pronounced and sustained increase in plasma glucose in both Angptl8and wild-type mice ( Figure 3 A). Plasma insulin levels were increased >6-fold in both Angptl8and wild-type mice ( Figure 3 B). Circulating triglyceride levels were unchanged by S961 in both strains of mice (data not shown). Mice receiving S961 were severely glucose intolerant ( Figure 3 C), which was associated with a compensatory doubling of the beta cell area and mass in both Angptl8and wild-type mice ( Figures 3 D and S2 ). These data show that insulin receptor antagonism causes glucose intolerance and beta cell expansion by a mechanism that does not depend upon ANGPTL8.

Beta cell mass in Angptl8 −/− and WT mice treated with S961 (20 nMol/week) or vehicle for 13 days. All groups had 7-10 animals. ∗∗ p < 0.001. Values are mean ± SEM.

All groups had seven to ten animals. ∗∗ p < 0.001; ∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.0001. Values are mean ± SEM.

(D) Beta cell area as a percentage of total pancreas area in Angptl8and WT mice treated with S961 (20 nMol/week) or vehicle for 13 days. See also Figure S2

(C) Oral glucose tolerance test in Angptl8 −/− and WT mice treated with S961 (20 nMol/week) or vehicle at day 11 of the study.

(B) Nonfasted plasma insulin on days 0 and 13 in Angptl8 −/− and WT mice treated with S961 (20 nMol/week) or vehicle.

(A) Nonfasted plasma glucose in Angptl8 −/− and WT mice continuously treated with the insulin receptor antagonist S961 (20 nMol/week) or vehicle for 13 days.

Hydrodynamic delivery of ANGPTL8 expression construct to the livers of chow-fed mice resulted in high circulating ANGPTL8 levels ( Figure 2 A). Overexpression of ANGPTL8 for 8 days significantly increased plasma triglyceride levels ( Figure 2 B) but had no effect on plasma glucose or insulin concentrations ( Figures 2 C and 2D). Oral glucose tolerance tests revealed no difference between control and ANGPTL8 overexpressing mice ( Figure 2 E). Beta cell area and mass were similar in control and ANGPTL8 overexpressing mice ( Figures 2 F and Figure S1 available online). Consistent with the data in Angptl8mice, these overexpression data do not support a role for ANGPTL8 in the regulation of beta cell function or number but confirm its involvement in triglyceride metabolism.

Beta cell mass in mice injected with control or ANGPTL8 DNA 8 days after HDD. The groups consisted of 6 (control) and 7 (ANGPTL8) animals. Values are mean ± SEM.

All groups had six to seven animals. ∗∗∗∗ p < 0.0001. Values are mean ± SEM. The experiment was repeated and the results were similar.

(F) Beta cell area as a percentage of total pancreas area in mice injected with control or ANGPTL8 DNA 8 days after HDD. See also Figure S1

(D) Nonfasted plasma insulin in mice injected with control or ANGPTL8 DNA before (day 0) and 8 days after HDD.

(C) Nonfasted plasma glucose in mice injected with control or ANGPTL8 DNA before (day 0) and 8 days after HDD.

(B) Nonfasted plasma triglyceride levels in mice injected with control or ANGPTL8 DNA before (day 0) and 8 days after HDD.

(A) Western blot analysis of plasma following hydrodynamic delivery (HDD) via tail vein injection of cDNA encoding a C-terminal V5 epitope-tagged human ANGPTL8. The control animals were injected with empty vector. An anti-V5 antibody was used for detection of ANGPTL8.

Mice lacking ANGPTL8 had normal nonfasted plasma glucose and insulin levels, but only one-third of the circulating triglycerides of wild-type mice ( Figures 1 A–1C), as reported previously (). In addition, we confirmed that Angptl8mice had normal glycemic control as revealed by an oral glucose tolerance ( Figure 1 E) and insulin tolerance tests ( Figure 1 F) (). Angptl8and wild-type mice fed a high-fat diet (60% kCal fat) for 8 weeks had a 40% increase in body weight ( Figure 1 D), which was associated with impaired glucose tolerance ( Figure 1 E) and reduced insulin sensitivity ( Figure 1 F). No differences in the induction of impaired glucose tolerance or insulin resistance were observed between wild-type and knock-out mice. Moreover, the increase in beta cell area (stained for insulin in brown) in fat-fed mice was comparable in Angptl8and wild-type mice ( Figures 1 G and 1H). These data indicate that ANGPTL8 is not essential for beta cell number or function under normal conditions and is not required for the compensatory beta cell expansion in response to the insulin resistance resulting from high-fat diet.

(H) Beta cell area as a percentage of total pancreas area. All groups had five to seven animals. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Values are mean ± SEM. The experiment was repeated and the results were similar.

(D) Body weight in WT and Angptl8 −/− mice on chow diet and following 8 weeks on high-fat diet (HFD).

(C) Plasma triglyceride levels in WT and Angptl8 −/− mice on chow diet after 18 hr fasting and 6 hr refeeding.

Discussion

The main findings of the study are that (1) ANGPTL8 is not required for beta cell function or the compensatory beta cell growth response to insulin resistance, (2) overexpression of ANGPTL8 does not increase beta cell area nor improve glycemic control, and (3) ANGPTL8 regulates plasma triglyceride levels.

Yi et al. (2013) Yi P.

Park J.-S.

Melton D.A. Betatrophin: a hormone that controls pancreatic β cell proliferation. Jiao et al., 2014 Jiao Y.

Le Lay J.

Yu M.

Naji A.

Kaestner K.H. Elevated mouse hepatic betatrophin expression does not increase human β-cell replication in the transplant setting. −/− and wild-type mice. Thus, ANGPTL8 is not required for the expansion of the beta cell area in response to insulin resistance induced by the insulin receptor antagonist S961. reported that ANGPTL8 is a betatrophin that controls beta cell proliferation in mice treated with the insulin receptor antagonist S961. A subsequent publication () and our data confirm the findings that administration of S961 causes severe glucose intolerance and beta cell expansion in mice. However, the beta cell growth response was similar in Angptl8and wild-type mice. Thus, ANGPTL8 is not required for the expansion of the beta cell area in response to insulin resistance induced by the insulin receptor antagonist S961.

−/− mice show no difference in glucose homeostasis ( Wang et al., 2013 Wang Y.

Quagliarini F.

Gusarova V.

Gromada J.

Valenzuela D.M.

Cohen J.C.

Hobbs H.H. Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis. −/− mice can mount a significant and similar expansion of their beta cell area in response to high-fat diet-induced insulin resistance. The lack of involvement of ANGPTL8 in the regulation of beta cell function and growth is supported by our hydrodynamic overexpression studies. Specifically, high circulating levels of ANGPTL8 in this study were associated with an increase in plasma triglycerides but caused no change in glycemic control or increase in beta cell area. The lack of expansion of the beta cell area could theoretically be due to simultaneous increases in replication and apoptosis frequencies. However, even if this were the case, it would not change our observation that Angptl8 overexpression did not increase beta cell area, contrary to what was reported by Yi et al. (2013) Yi P.

Park J.-S.

Melton D.A. Betatrophin: a hormone that controls pancreatic β cell proliferation. It had previously been reported that wild-type and Angptl8mice show no difference in glucose homeostasis (). In the current study, we demonstrate that both wild-type and Angptl8mice can mount a significant and similar expansion of their beta cell area in response to high-fat diet-induced insulin resistance. The lack of involvement of ANGPTL8 in the regulation of beta cell function and growth is supported by our hydrodynamic overexpression studies. Specifically, high circulating levels of ANGPTL8 in this study were associated with an increase in plasma triglycerides but caused no change in glycemic control or increase in beta cell area. The lack of expansion of the beta cell area could theoretically be due to simultaneous increases in replication and apoptosis frequencies. However, even if this were the case, it would not change our observation that Angptl8 overexpression did not increase beta cell area, contrary to what was reported by

Espes et al., 2014 Espes D.

Lau J.

Carlsson P.-O. Increased circulating levels of betatrophin in individuals with long-standing type 1 diabetes. Fu et al., 2014 Fu Z.

Berhane F.

Fite A.

Seyoum B.

Abou-Samra A.B.

Zhang R. Elevated circulating lipasin/betatrophin in human type 2 diabetes and obesity. Fenzl et al., 2014 Fenzl A.

Itariu B.K.

Kosi L.

Fritzer-Szekeres M.

Kautzky-Willer A.

Stulnig T.M.

Kiefer F.W. Circulating betatrophin correlates with atherogenic lipid profiles but not with glucose and insulin levels in insulin-resistant individuals. Peloso et al., 2014 Peloso G.M.

Auer P.L.

Bis J.C.

Voorman A.

Morrison A.C.

Stitziel N.O.

Brody J.A.

Khetarpal S.A.

Crosby J.R.

Fornage M.

et al. NHLBI GO Exome Sequencing Project

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks. Studies in humans suggest that ANGPTL8 levels are increased in patients with type 1 diabetes. However, ANGPTL8 levels do not correlate with residual C-peptide levels, glucose control, or insulin requirement, suggesting that ANGPTL8 does not protect against beta cell destruction (). The role of ANGPTL8 in type 2 diabetes and obesity remains controversial. ANGPTL8 levels were found in one study to correlate with type 2 diabetes and body mass index (), whereas another study reported no difference in ANGPTL8 levels between lean and morbidly obese or between nondiabetic or type 2 diabetic individuals. In the latter study, ANGPTL8 levels were associated with an atherogenic lipid profile (). Genetic sequencing of 56,000 individuals revealed a low-frequency coding DNA sequence variant in ANGPTL8 that was associated with higher plasma HDL-cholesterol and lower triglycerides but not with fasting glucose levels (), once again confirming the role of ANGPTL8 in lipid metabolism rather than in regulation of human beta cell function.

In conclusion, the present data do not support a role for ANGPTL8 in the control of beta cell function or growth. The findings that plasma triglyceride levels are markedly reduced by Angptl8 deletion and increased following ANGPTL8 overexpression support the possibility that inhibition of ANGPTL8 represents a therapeutic strategy for hypertriglyceridemia.